Conjugates comprising peptide groups and methods related thereto

ABSTRACT

In some aspects, the invention relates to an antibody-drug conjugate, comprising an antibody; a linker; and at least two active agents. In preferred embodiments, the linker comprises a peptide sequence of a plurality of amino acids, and at least two of the active agents are covalently coupled to side chains of the amino acids. The antibody-drug conjugate may comprise a self-immolative group, preferably two-self-immolative groups. The linker may comprise an O-substituted oxime, e.g., wherein the oxygen atom of the oxime is substituted with a group that covalently links the oxime to the active agent; and the carbon atom of the oxime is substituted with a group that covalently links the oxime to the antibody.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is the U.S. 371(c) national stage application ofPCT/IB2016/001810, filed Nov. 23, 2016, which claims the benefit ofpriority to U.S. Provisional Application Ser. No. 62/259,997, filed Nov.25, 2015, and U.S. Provisional Application Ser. No. 62/260,006, filedNov. 25, 2015, each of which is hereby incorporated by reference in itsentirety.

BACKGROUND

Antibody-drug conjugate (ADC) technology is a target-orientedtechnology, which allows for selective apoptosis of cancer cells.Typically, ADCs function by targeting cancer cells using the antibodyand then releasing a toxic material (i.e., the drug) in a cell, therebytriggering cell death. Since ADC technology allows a drug to beaccurately delivered to a target cancer cell and released under specificconditions, while minimizing collateral damage to healthy cells, ADCtechnology increases the efficacy of a therapeutic antibody anddecreases the risk of an adverse reaction.

Recently, a new approach to making antibody-drug conjugates has beendescribed, using protein prenylation of a C-terminal amino acid sequenceto install a modified isoprenoid unit that allows for attachment of adrug or other active agent to the antibody in a mild and site-specificmanner (e.g., see U.S. Patent Publication No. 2012/0308584). Furtherrefinement is possible.

Improved strategies for configuring ADCs remain desirable.

SUMMARY

In some aspects, the invention relates to an antibody-drug conjugate,comprising an antibody; a linker; and at least two active agents. Inpreferred embodiments, the linker comprises a peptide sequence of aplurality of amino acids, and at least two of the active agents arecovalently coupled to side chains of the amino acids. The antibody-drugconjugate may comprise a self-immolative group, preferablytwo-self-immolative groups. The linker may comprise an O-substitutedoxime, e.g., wherein the oxygen atom of the oxime is substituted with agroup that covalently links the oxime to the active agents; and thecarbon atom of the oxime is substituted with a group that covalentlylinks the oxime to the antibody.

In some aspects, the invention relates to an antibody-drug conjugate,comprising an antibody; a linker covalently coupled to the antibody; andat least two active agents covalently coupled to the linker. The linkermay comprise a peptide sequence of a plurality of amino acids. At leasttwo of the active agents may be covalently coupled to side chains of theamino acids.

At least two of the active agents may be covalently coupled to sidechains of lysine, 5-hydroxylysine, 4-oxalysine, 4-thialysine,4-selenalysine, 4-thiahomolysine, 5,5-dimethyllysine,5,5-difluorolysine, trans-4-dehydrolysine, 2,6-diamino-4-hexynoic acid,cis-4-dehydrolysine, 6-N-methyllysine, diaminopimelic acid, ornithine,3-methylornithine, α-methylornithine, citrulline, and/or homocitrulline.For example, at least two of the active agents may be covalently coupledto side chains of lysine or ornithine.

The plurality of amino acids may comprise L-amino acids or D-aminoacids. The plurality of amino acids may comprise α-amino acids orβ-amino acids. The plurality of amino acids may comprise alanine,aspartate, asparagine, glutamate, glutamine, glycine, histidine, lysine,ornithine, proline, serine, and/or threonine. In certain preferredembodiments, the peptide does not comprise isoleucine, methionine,leucine, phenylalanine, tryptophan, tyrosine, or valine. The peptide maycomprise 2 to 20 amino acids.

In preferred embodiments, the N-terminus or the C-terminus of thepeptide is substituted with a group that covalently links at least oneof the active agents to the peptide. In preferred embodiments, theN-terminus or the C-terminus of the peptide is substituted with a groupthat covalently links the antibody to the peptide.

In some embodiments, the linker comprises an O-substituted oxime; theoxygen atom of the oxime is substituted with a group that covalentlylinks the oxime to the peptide; and the carbon atom of the oxime issubstituted with a group that covalently links the oxime to theantibody. In some embodiments, the linker comprises an O-substitutedoxime; the carbon atom of the oxime is substituted with a group thatcovalently links the oxime to the peptide; and the oxygen atom of theoxime is substituted with a group that covalently links the oxime to theantibody.

In some embodiments, the linker comprises an alkylene having 1 to 100carbon atoms. The alkylene may comprise at least one unsaturated bond.In preferred embodiments, the alkylene comprises at least one carbonatom of the alkylene is replaced by one or more heteroatoms selectedfrom nitrogen (N), oxygen (O), and sulfur (S). In certain embodiments,the alkylene is further substituted with at least one alkyl having 1 to20 carbon atoms.

In preferred embodiments, the linker is covalently bound to the antibodyby a thioether bond, and the thioether bond comprises a sulfur atom of acysteine of the antibody.

In some embodiments, the antibody comprises a C-terminal amino acidmotif that is recognized by an isoprenoid transferase; and the thioetherbond comprises a sulfur atom of a cysteine of the amino acid motif.

In some embodiments, the antibody comprises an amino acid motif and theamino acid motif is a sequence selected from CXX, CXC, XCXC, XXCC, andCYYX, wherein C represents cysteine; Y, independently for eachoccurrence, represents an aliphatic amino acid; and X, independently foreach occurrence, represents glutamine, glutamate, serine, cysteine,methionine, alanine, or leucine. In certain preferred embodiments, thelinker is covalently bound to the antibody by a thioether bond thatcomprises a sulfur atom of a cysteine of the amino acid motif. The aminoacid motif may be the sequence CYYX; wherein Y, independently for eachoccurrence, represents alanine, isoleucine, leucine, methionine, orvaline. For example, the amino acid motif may be CVIM or CVLL. Inpreferred embodiments, at least one of the seven amino acids precedingthe amino acid motif is glycine.

In some embodiments, at least three of the seven amino acids precedingthe amino acid motif are each independently selected from glycine andproline. In certain preferred embodiments, one to ten amino acidspreceding the amino acid motif may be glycine, preferably at least five(e.g., GGGGG), such as where each of the five, six, seven, eight, nine,or ten amino acids preceding the amino acid motif is glycine. In certainpreferred embodiments, at least three of the seven amino acids precedingthe amino acid motif are each independently selected from glycine,aspartic acid, arginine, and serine.

In certain preferred embodiments, a C-terminus of the antibody comprisesthe amino acid sequence GGGGGGGCVIM.

In some embodiments, the linker is covalently bound to the antibody by athioether bond, and the thioether bond comprises a carbon atom of atleast one isoprenyl unit, represented by

e.g., wherein n is at least 2. The linker may comprise an oxime, and theat least one isoprenyl unit covalently links the oxime to the antibody.

In some embodiments, the linker may comprise

In some embodiments, the linker comprises:

In some embodiments, the linker comprises at least one polyethyleneglycol unit, represented by

In some embodiments, the linker comprises linker comprises 1 to 20—OCH₂CH₂-units. For example, the linker may comprise 4 to 20 —OCH₂CH₂—units. In certain preferred embodiments, the linker may comprise 1 to 12—OCH₂CH₂— units. In other preferred embodiments, the linker may comprise3 to 12 —OCH₂CH₂— units. In some embodiments, the linker comprises anoxime, and the at least one polyethylene glycol unit covalently linksthe oxime to the peptide.

In some embodiments, the linker comprises a connection unit representedby —(CH₂)_(r)(V(CH₂)_(p))_(q)—, wherein:

-   -   r is an integer from 0 to 10, preferably 2;    -   p is an integer from 0 to 12, preferably 2;    -   q is an integer from 1 to 20;    -   V is a single bond, —O—, —S—, —NR₂₁—, —C(O)NR₂₂—, —NR₂₃C(O)—,        —NR₂₄SO₂—, or —SO₂NR₂₅—, preferably —O—; and    -   R₂₁ to R₂₅ are each independently hydrogen, (C₁-C₆)alkyl,        (C₁-C₆)alkyl(C₆-C₂₀)aryl, or (C₁-C₆)alkyl(C₃-C₂₀)heteroaryl.

In some embodiments, the linker comprises a connection unit representedby —(CH₂)_(r)(V(CH₂)_(p))_(q)—, —((CH₂)_(p)V)_(q)—,—(CH₂)_(r)(V(CH₂)_(p))_(q)—, —((CH₂)_(p)V)_(q)(CH₂)_(r)—,—Y(((CH₂)_(p)V)_(q)— or —(CH₂)_(r)(V(CH₂)_(p))_(q)YCH₂—

wherein:

-   -   r is an integer from 0 to 10;    -   p is an integer from 1 to 10;    -   q is an integer from 1 to 20;    -   V and Y are each independently a single bond, —O—, —S—, —NR₂₁—,        —C(O)NR₂₂—, —NR₂₃C(O)—, —NR₂₄SO₂—, or —SO₂NR₂₅—; and    -   R₂₁ to R₂₅ are each independently hydrogen, (C₁-C₆)alkyl,        (C₁-C₆)alkyl(C₆-C₂₀)aryl or (C₁-C₆)alkyl(C₃-C₂₀)heteroaryl.

In certain preferred embodiments of these linkers, q is an integer from4 to 20. In some embodiments q is an integer from 6 to 20. In otherpreferred embodiments, q is an integer from 2 to 12. In someembodiments, q is 2, 5 or 11.

In some embodiments of these linkers, r is 2. In some embodiments ofthese linkers, p is 2. In some embodiments, V and Y are eachindependently —O—. In some embodiments, r is 2, p is 2, q is 2, 5, or11, and V is —O—.

In some embodiments, the linker comprises a connection unit representedby —(CH₂CH₂X)_(w)—, wherein:

-   -   X represents —O—, (C₁-C₈)alkylene, or —NR₂₁—, preferably —O—;    -   R₂₁ represents hydrogen, (C₁-C₆)alkyl, (C₁-C₆)alkyl(C₆-C₂₀)aryl,        or (C₁-C₆)alkyl(C₃-C₂₀)heteroaryl, preferably hydrogen; and    -   w is an integer from 1 to 20, preferably 4 to 12.

In some embodiments, the linker comprises a binding unit formed by a1,3-dipolar cycloaddition reaction, hetero-Diels-Alder reaction,nucleophilic substitution reaction, non-aldol type carbonyl reaction,addition to carbon-carbon multiple bond, oxidation reaction, or clickreaction. For example, a binding unit may be formed by a reactionbetween acetylene and azide, or a reaction between an aldehyde or ketonegroup and a hydrazine or alkoxyamine.

A binding unit may be represented by any one of Formulas A, B, C, or D,preferably C or D:

-   -   wherein:    -   L₁ is a single bond or alkylene having 1 to 30 carbon atoms,        preferably 12;    -   R₁₁ is hydrogen or alkyl having 1 to 10 carbon atoms, preferably        methyl; and    -   L₂ is alkylene having 1 to 30 carbon atoms, e.g., 10 or 11,        preferably 11.

In some embodiments, the linker comprises

wherein

-   -   V is a single bond, —O—, —S—, —NR₂₁—, —C(O)NR₂₂—, —NR₂₃C(O)—,        —NR₂₄SO₂—, or —SO₂NR₂₅—, preferably —O—;    -   R₂₁ to R₂₅ are each independently hydrogen, (C₁-C₆)alkyl,        (C₁-C₆)alkyl(C₆-C₂₀)aryl, or (C₁-C₆)alkyl(C₃-C₂₀)heteroaryl;    -   r is an integer from 1 to 10, preferably 3;    -   p is an integer from 0 to 10, preferably 2;    -   q is an integer from 1 to 20, preferably 2 to 20; and    -   L₁ is a single bond.

In some embodiments, the antibody-drug conjugate comprises the structureof Formula V:

-   -   wherein:    -   A represents the antibody;    -   P represents the peptide    -   B₁ represents a first active agent;    -   B₂ represents a second active agent; and    -   n is an integer from 1 to 20.

In some embodiments, the antibody-drug conjugate comprises the structureof Formula VI:

-   -   wherein    -   A represents the antibody;    -   P represents the peptide;    -   B₁ represents a first active agent;    -   B₂ represents a second active agent; and    -   n is an integer from 1 to 20.

In some embodiments, the conjugate comprises a structure of the formula:

-   -   wherein    -   A represents a ligand;    -   P        P represents the peptide;    -   B₁ represents a first active agent;    -   B₂ represents a second active agent;    -   L₁ represents a linker, optionally including a cleavage group;    -   L₂ represents a linker, optionally including a cleavage group;        and    -   n is an integer from 1 to 20.

In some embodiments, the conjugate comprises a structure of the formula:

-   -   wherein    -   A represents a ligand;    -   P        P represents the peptide;    -   B₁ represents a first active agent;    -   B₂ represents a second active agent;    -   B₃ represents a third active agent;    -   L₁ represents a linker, optionally including a cleavage group;    -   L₂ represents a linker, optionally including a cleavage group;    -   L₃ represents a linker, optionally including a cleavage group;        and    -   n is an integer from 1 to 20.

In some embodiments, the conjugate comprises a structure of the formula:

-   -   wherein    -   A represents a ligand;    -   P        P represents the peptide;    -   B₁ represents a first active agent;    -   B₁₁ represents a second active agent;    -   B₁₂ represents a third active agent;    -   L₁ represents a linker;    -   L₂ represents a linker;    -   L₁₁ represents a secondary linker, optionally including a        cleavage group;    -   L₁₂ represents a secondary linker, optionally including a        cleavage group;    -   BU represents a branching unit; and    -   n is an integer from 1 to 20.

In some embodiments, the conjugate comprises a structure of the formula:

-   -   wherein    -   A represents a ligand;    -   P        P represents the peptide;    -   B₁ represents a first active agent;    -   B₂ represents a second active agent;    -   L₁ represents a linker, optionally including a cleavage group;    -   L₂ represents a linker, optionally including a cleavage group;        and    -   n is an integer from 1 to 20.

In some embodiments, the conjugate comprises a structure of the formula:

-   -   wherein    -   A represents a ligand;    -   P        P represents the peptide;    -   B₁ represents a first active agent;    -   B₂ represents a second active agent;    -   B₃ represents a third active agent;    -   L₁ represents a linker, optionally including a cleavage group;    -   L₂ represents a linker, optionally including a cleavage group;    -   L₃ represents a linker, optionally including a cleavage group;        and    -   n is an integer from 1 to 20.

In some embodiments, the conjugate comprises a structure of the formula:

-   -   wherein    -   A represents a ligand;    -   P        P represents the peptide;    -   B₁ represents a first active agent;    -   B₁₁ represents a second active agent;    -   B₁₂ represents a third active agent;    -   L₁ represents a linker;    -   L₂ represents a linker;    -   L₁₁ represents a secondary linker, optionally including a        cleavage group;    -   L₁₂ represents a secondary linker, optionally including a        cleavage group;    -   BU represents a branching unit; and    -   n is an integer from 1 to 20.

In certain preferred embodiments, the ligand is an antibody. Theantibody may be a monoclonal antibody, polyclonal antibody, antibodyfragment, Fab, Fab′, Fab′-SH, F(ab′)₂, Fv, single chain Fv (“scFv”),diabody, linear antibody, bispecific antibody, multispecific antibody,chimeric antibody, humanized antibody, human antibody, or fusion proteincomprising the antigen-binding portion of an antibody.

The antibody may be selected from muromonab-CD3 abciximab, daclizumab,palivizumab, infliximab, trastuzumab, etanercept, basiliximab,gemtuzumab, alemtuzumab, ibritumomab, adalimumab, alefacept, omalizumab,efalizumab, tositumomab, cetuximab, ABT-806, bevacizumab, natalizumab,ranibizumab, panitumumab, eculizumab, rilonacept, certolizumab,romiplostim, AMG-531, golimumab, ustekinumab, ABT-874, belatacept,belimumab, atacicept, an anti-CD20 antibody, canakinumab, tocilizumab,atlizumab, mepolizumab, pertuzumab, HuMax CD20, tremelimumab,ticilimumab, ipilimumab, IDEC-114, inotuzumab, HuMax EGFR, aflibercept,HuMax-CD4, teplizumab, otelixizumab, catumaxomab, the anti-EpCAMantibody IGN101, adecatumomab, oregovomab, dinutuximab, girentuximab,denosumab, bapineuzumab, motavizumab, efumgumab, raxibacumab, ananti-CD20 antibody, LY2469298, and veltuzumab.

In some embodiments, each active agent is independently selected fromchemotherapeutic agents and toxins.

Each active agent may be independently selected from: (a) erlotinib,bortezomib, fulvestrant, sutent, letrozole, imatinib mesylate, PTK787/ZK222584, oxaliplatin, 5-fluorouracil, leucovorin, rapamycin, lapatinib,lonafarnib, sorafenib, gefitinib, AG1478, AG1571, thiotepa,cyclophosphamide, busulfan, improsulfan, piposulfan, benzodopa,carboquone, meturedopa, uredopa, ethylenimine, altretamine,triethylenemelamine, trietylenephosphoramide,triethiylenethiophosphoramide, trimethylolomelamine, bullatacin,bullatacinone, camptothecin, topotecan, bryostatin, callystatin,CC-1065, adozelesin, carzelesin, bizelesin, cryptophycin 1, cryptophycin8, dolastatin, duocarmycin, KW-2189, CB1-TM1, eleutherobin,pancratistatin, sarcodictyin, spongistatin, chlorambucil,chlornaphazine, cholophosphamide, estramustine, ifosfamide,mechlorethamine, melphalan, novembichin, phenesterine, prednimustine,trofosfamide, uracil mustard, carmustine, chlorozotocin, fotemustine,lomustine, nimustine, ranimnustine, calicheamicin, calicheamicin gamma1, calicheamicin omega 1, dynemicin, dynemicin A, clodronate,esperamicin, neocarzinostatin chromophore, aclacinomysins, actinomycin,antrmycin, azaserine, bleomycins, cactinomycin, carabicin, carninomycin,carzinophilin, chromomycins, dactinomycin, daunorubicin, detorubucin,6-diazo-5-oxo-L-norleucine, doxorubicin, morpholino-doxorubicin,cyanomorpholino-doxorubicin, 2-pyrrolino-doxorubucin, liposomaldoxorubicin, deoxydoxorubicin, epirubicin, esorubicin, marcellomycin,mitomycin C, mycophenolic acid, nogalamycin, olivomycins, peplomycin,potfiromycin, puromycin, quelamycin, rodorubicin, streptomigrin,streptozocin, tubercidin, ubenimex, zinostatin, zorubicin,5-fluorouracil, denopterin, methotrexate, pteropterin, trimetrexate,fludarabine, 6-mercaptopurine, thiamiprine, thiguanine, ancitabine,azacitidine, 6-azauridine, carmofur, cytarabine, dideoxyuridine,doxifluridine, enocitabine, floxuridine, calusterone, dromostanolonepropionate, epitiostanol, mepitiostane, testolactone, aminoglutethimide,mitotane, trilostane, folinic acid, aceglatone, aldophosphamideglycoside, aminolevulinic acid, eniluracil, amsacrine, bestrabucil,bisantrene, edatraxate, defofamine, demecolcine, diaziquone,elfornithine, elliptinium acetate, etoglucid, gallium nitrate,hydroxyurea, lentinan, lonidainine, maytansine, ansamitocins,mitoguazone, mitoxantrone, mopidanmol, nitraerine, pentostatin,phenamet, pirarubicin, losoxantrone, 2-ethylhydrazide, procarbazine,polysaccharide-k, razoxane, rhizoxin, sizofiran, spirogermanium,tenuazonic acid, triaziquone, 2,2′,2″-trichlorotriethylamine, T-2 toxin,verracurin A, roridin A, and anguidine, urethane, vindesine,dacarbazine, mannomustine, mitobronitol, mitolactol, pipobroman,gacytosine, arabinoside, cyclophosphamide, thiotepa, paclitaxel,albumin-engineered nanoparticle formulation of paclitaxel, doxetaxel,chlorambucil, gemcitabine, 6-thioguanine, mercaptopurine, cisplatin,carboplatin, vinblastine, platinum, etoposide, ifosfamide, mitoxantrone,vincristine, vinorelbine, novantrone, teniposide, edatrexate,daunomycin, aminopterin, xeloda, ibandronate, CPT-11, topoisomeraseinhibitor RFS 2000, difluoromethylomithine, retinoic acid, capecitabine,or pharmaceutically acceptable salts, solvates or acids of any of theforegoing;

(b) monokine, a lymphokine, a traditional polypeptide hormone,parathyroid hormone, thyroxine, relaxin, prorelaxin, a glycoproteinhormone, follicle stimulating hormone, thyroid stimulating hormone,luteinizing hormone, hepatic growth factor fibroblast growth factor,prolactin, placental lactogen, tumor necrosis factor-α, tumor necrosisfactor-β, mullerian-inhibiting substance, mouse gonadotropin-associatedpeptide, inhibin, activin, vascular endothelial growth factor,thrombopoietin, erythropoietin, an osteoinductive factor, an interferon,interferon-α, interferon-β, interferon-γ, a colony stimulating factor(“CSF”), macrophage-CSF, granulocyte-macrophage-CSF, granulocyte-CSF, aninterleukin (“IL”), IL-1, IL-1α, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7,IL-8, IL-9, IL-10, IL-11, IL-12, a tumor necrosis factor, TNF-α, TNF-β,a polypeptide factor, LIF, kit ligand, or a combination of any of theforegoing;

(c) diphtheria toxin, botulium toxin, tetanus toxin, dysentery toxin,cholera toxin, amanitin, α-amanitin, amanitin derivatives,pyrrolobenzodiazepine, pyrrolobenzodiazepine derivatives, tetrodotoxin,brevetoxin, ciguatoxin, ricin, AM toxin, tubulysin, geldanamycin,maytansinoid, calicheamicin, daunomycin, doxorubicin, methotrexate,vindesine, SG2285, dolastatin, a dolastatin analog, auristatin,cryptophycin, camptothecin, camptothecin derivatives and metabolites(e.g., SN-38), rhizoxin, a rhizoxin derivative, CC-1065, a CC-1065analogue or derivative, duocarmycin, an enediyne antibiotic,esperamicin, epothilone, azonafide, aplidine, a toxoid, or a combinationof any of the foregoing;

(d) an affinity ligand, wherein the affinity ligand is a substrate, aninhibitor, a stimulating agent, a neurotransmitter, a radioisotope, or acombination of any of the foregoing;

(e) a radioactive label, ³²P, ³⁵S, a fluorescent dye, an electron densereagent, an enzyme, biotin, streptavidin, dioxigenin, a hapten, animmunogenic protein, a nucleic acid molecule with a sequencecomplementary to a target, or a combination of any of the foregoing;

(f) an immunomodulatory compound, an anti-cancer agent, an anti-viralagent, an anti-bacterial agent, an anti-fungal agent, and ananti-parasitic agent, or a combination of any of the foregoing;

(g) tamoxifen, raloxifene, droloxifene, 4-hydroxytamoxifen, trioxifene,keoxifene, LY117018, onapristone, or toremifene;

(h) 4(5)-imidazoles, aminoglutethimide, megestrol acetate, exemestane,letrozole, or anastrozole;

(i) flutamide, nilutamide, bicalutamide, leuprolide, goserelin, ortroxacitabine;

(j) an aromatase inhibitor;

(k) a protein kinase inhibitor;

(l) a lipid kinase inhibitor;

(m) an antisense oligonucleotide;

(n) a ribozyme;

(o) a vaccine; and

(p) an anti-angiogenic agent.

In some embodiments, the at least one active agent is taltobulin orazonafide.

In some embodiments, the conjugate comprises a moiety selected from:

In certain embodiments, each active agent is independently selected fromamanitin, auristatin, calicheamicin, camptothecin, cryptophycin,daunomycin, dolastatin, doxorubicin, duocarmycin, epothilone,esperamicin, geldanamycin, maytansinoid, methotrexate, monomethylauristatin E (“MMAE”), monomethyl auristatin F (“MMAF”),pyrrolobenzodiazepine, rhizoxin, SG2285, tubulysin, vindesine, andtoxoid, or a derivative of any one of the foregoing. For example, eachactive agent may be independently selected from amanitin, MMAE, andMMAF, or a derivative of any one of the foregoing.

In preferred embodiments, at least one active agent is coupled to thelinker through a cleavage group, e.g., a group having the structure ofFormula (I):

wherein:

-   -   B represents an active agent;    -   G represents a sugar or sugar acid, preferably glucuronic acid;    -   W represents an electron-withdrawing group, preferably        —C(O)NR′—, where C(O) is bonded to the phenyl ring and NR′ is        bonded to L;    -   each Z independently represents hydrogen, (C₁-C₈)alkyl, or an        electron-withdrawing group (such as an amide, carboxylic acid,        carboxylic acid ester, halogen, cyano, or nitro), preferably a        hydrogen, (C₁-C₈)alkyl, halogen, cyano, or nitro, most        preferably hydrogen;    -   n is an integer from 1 to 3, preferably 3;    -   R₁ and R₂ are each independently hydrogen, (C₁-C₈)alkyl, or        (C₃-C₈)cycloalkyl, preferably hydrogen, or R₁ and R₂ taken        together with the carbon atom to which they are attached form a        (C₃-C₈)cycloalkyl ring; and    -   L represents a linkage to the antibody.

In some embodiments, at least one active agent is coupled to the linkerthrough a cleavage group having the formula:

-   wherein:-   G represents a sugar, sugar acid, or modified sugar, preferably a    sugar or sugar acid, most preferably glucuronic acid;-   W represents —C(O)—, —C(O)NR′—, —C(O)O—, —S(O)₂NR′—, —P(O)R″NR′—,    —S(O)NR′—, or —PO₂NR′—, in each case where the C(O), S, or P is    preferably directly bound to the phenyl ring, and R′ and R″ are each    independently hydrogen, (C₁-C₈)alkyl, mono- or    di-carboxyl(C₁-C₈)alkyl, (C₃-C₈)cycloalkyl, (C₁-C₈)alkoxy,    (C₁-C₈)alkylthio, mono- or di-(C₁-C₈)alkylamino, (C₃-C₂₀)heteroaryl,    or (C₆-C₂₀)aryl;-   each Z independently represents hydrogen, (C₁-C₈)alkyl, or an    electron-withdrawing group (such as an amide, carboxylic acid,    carboxylic acid ester, halogen, cyano, or nitro), preferably a    hydrogen, (C₁-C₈)alkyl, halogen, cyano, or nitro, most preferably    hydrogen;-   n is an integer from 1 to 3;-   m is 0 or 1, preferably 1; and-   R₁ and R₂ are each independently hydrogen, (C₁-C₈)alkyl, or    (C₃-C₈)cycloalkyl, preferably hydrogen, or R₁ and R₂ taken together    with the carbon atom to which they are attached form a    (C₃-C₈)cycloalkyl ring.

In some embodiments, at least one active agent is coupled to the linkerthrough a cleavage group having the formula:

-   or a pharmaceutically acceptable salt thereof, wherein-   G represents a sugar, sugar acid, or modified sugar, preferably a    sugar or sugar acid, most preferably glucuronic acid;-   B is a unit covalently attached to the active agent,-   W represents —C(O)—, —C(O)NR′—, —C(O)O—, —S(O)₂NR′—, —P(O)R″NR′—,    —S(O)NR′—, or —PO₂NR′—, in each case where the C(O), S, or P is    directly bound to the phenyl ring, and R′ and R″ are each    independently hydrogen, (C₁-C₈)alkyl, (C₃-C₈)cycloalkyl,    (C₁-C₈)alkoxy, (C₁-C₈)alkylthio, mono- or di-(C₁-C₈)alkylamino,    (C₃-C₂₀)heteroaryl, or (C₆-C₂₀)aryl;-   each Z independently represents hydrogen, (C₁-C₈)alkyl, or an    electron-withdrawing group (such as an amide, carboxylic acid,    carboxylic acid ester, halogen, cyano, or nitro), preferably a    hydrogen, (C₁-C₈)alkyl, halogen, cyano, or nitro, most preferably    hydrogen;-   n is an integer from 1 to 3, preferably 3;-   L represents a linker comprising the peptide sequence;-   R₁ and R₂ are each independently hydrogen, (C₁-C₈)alkyl, or    (C₃-C₈)cycloalkyl, preferably hydrogen, or R₁ and R₂ taken together    with the carbon atom to which they are attached form a    (C₃-C₈)cycloalkyl ring.

The sugar or sugar acid may be, for example, a monosaccharide. In someembodiments,

G is

R₃ is hydrogen or a carboxyl protecting group; and

each R₄ is independently hydrogen or a hydroxyl protecting group.

For example, R₃ may be hydrogen and each R₄ may be hydrogen.

W may be —C(O)—, —C(O)NR′—, —C(O)O—, —S(O)₂NR′—, —P(O)R″NR′—, —S(O)NR′—,or —PO₂NR′—, in each case where the C(O), S, or P is directly bound tothe phenyl ring, and R′ and R″ are each independently hydrogen,(C₁-C₈)alkyl, (C₃-C₈)cycloalkyl, (C₁-C₈)alkoxy, (C₁-C₈)alkylthio, mono-or di-(C₁-C₈)alkylamino, (C₃-C₂₀)heteroaryl, or (C₆-C₂₀)aryl. Inpreferred embodiments, W represents —C(O)NR′—, where C(O) is bonded tothe phenyl ring and NR′ is bonded to L.

Alternative cleavage groups includevaline-citrulline-p-aminobenzylcarbamate (VC-PABC).

In preferred embodiments, Z represents hydrogen and n is 3.

In preferred embodiments, R₁ and R₂ each represent hydrogen.

In preferred embodiments, G represents glucuronic acid; W represents—C(O)NR′—, where C(O) is bonded to the phenyl ring and NR′ is bonded toL; each Z independently represents hydrogen; and R₁ and R₂ eachrepresent hydrogen.

B, B₁, and/or B₂ may be each independently selected from:

wherein y is an integer from 1 to 10.

In some embodiments, an antibody-drug conjugate comprises 2 to 4 linkersas set forth in any of the preceding claims, covalently coupled to theantibody, wherein each linker preferentially comprises a peptidesequence of a plurality of amino acids, and at least two active agentsare preferentially covalently coupled to side chains of the amino acidsof each linker. Each linker may be coupled to a C-terminus of theantibody (e.g., of the heavy and light chains of the antibody).

In various embodiments, each active agent may be the same active agent.Alternatively, an antibody-drug conjugate may comprise at least twodifferent active agents, such as where each linker bears a differentactive agent from the other linkers.

Structures and components of related antibody-drug conjugates aredisclosed in PCT/KR2015/005299, which is hereby incorporated byreference in its entirety, in particular for the chemical formulae andgeneric structures of antibody-drug conjugates, their component parts(e.g., linkers, cleavage groups, etc.), and their preparation and use asdisclosed therein. In certain preferred embodiments, the variousconjugates and other aspects of the present invention specificallyexclude the various structures and methods disclosed inPCT/KR2015/005299.

In some aspects, the invention relates to a pharmaceutical compositioncomprising an antibody-drug conjugate as described herein and apharmaceutically acceptable excipient. A pharmaceutical composition mayfurther comprise a therapeutically effective amount of chemotherapeuticagent.

In some aspects, the invention relates to a method of treating cancer ina subject, comprising administering a pharmaceutical composition asdescribed herein to the subject.

The subject may be a mammal. For example, the subject may be selectedfrom rodents, lagomorphs, felines, canines, porcines, ovines, bovines,equines, and primates. In preferred embodiments, the subject is a human.

In some aspects, the invention relates to a method for making anantibody-drug conjugate as described herein, comprising reacting abiomolecule with a prodrug. The biomolecule preferentially comprises anantibody and a ketone or aldehyde. The prodrug preferentially comprisesan alkoxyamine. The reaction may produce an oxime, thereby covalentlylinking the antibody to the prodrug.

The method may further comprise isoprenylating the antibody, therebyproducing the biomolecule. For example, the antibody may comprise anisoprenylation sequence, and isoprenylating the antibody may compriseincubating the antibody with an isoprenoid transferase and an isoprenoidtransferase substrate. The substrate preferentially comprises the ketoneor aldehyde. The isoprenoid transferase may be, for example,farnesyltransferase or geranylgeranyltransferase.

In some embodiments, the invention relates to a method for making anantibody-drug conjugate as described herein, comprising isoprenylatingan antibody. For example, the antibody may comprise an isoprenylationsequence, and isoprenylating the antibody may comprise incubating theantibody with an isoprenoid transferase and an isoprenoid transferasesubstrate. In preferred embodiments, the substrate comprises at leastone active agent as described herein.

In some embodiments, the invention relates to a linker-active agentcompound, to which a ligand (preferably an antibody) can be conjugatedto result in one of the conjugates of the invention. Accordingly, insuch linker-active agent compounds, i) the linker comprises a peptidesequence of a plurality of amino acids; and ii) at least two activeagents are covalently coupled to side chains of the amino acids. In somesuch embodiments, each active agent is coupled to the peptide sequenceby a cleavage group that can be hydrolyzed to release the active agentfrom the ligand-active agent compound. The cleavage group may have astructure of the formula:

wherein:

B represents the active agent;

G represents a sugar or sugar acid, preferably glucuronic acid;

W represents an electron-withdrawing group, preferably —C(O)NR′—, whereC(O) is bonded to the phenyl ring and NR′ is bonded to L;

each Z independently represents hydrogen, (C₁-C₈)alkyl, or anelectron-withdrawing group (such as an amide, carboxylic acid,carboxylic acid ester, halogen, cyano, or nitro), preferably a hydrogen,(C₁-C₈)alkyl, halogen, cyano, or nitro, most preferably hydrogen;

n is an integer from 1 to 3, preferably 3;

R₁ and R₂ are each independently hydrogen, (C₁-C₈)alkyl, or(C₃-C₈)cycloalkyl, preferably hydrogen, or R₁ and R₂ taken together withthe carbon atom to which they are attached form a (C₃-C₈)cycloalkylring; and

L represents a linkage to the peptide sequence.

In other embodiments, the cleavage group may have a structure of theformula:

-   wherein:-   G represents a sugar, sugar acid, or modified sugar, preferably a    sugar or sugar acid, most preferably glucuronic acid;-   W represents —C(O)—, —C(O)NR′—, —C(O)O—, —S(O)₂NR′—, —P(O)R″NR′—,    —S(O)NR′—, or —PO₂NR′—, in each case where the C(O), S, or P is    preferably directly bound to the phenyl ring, and R′ and R″ are each    independently hydrogen, (C₁-C₈)alkyl, mono- or    di-carboxyl(C₁-C₈)alkyl, (C₃-C₈)cycloalkyl, (C₁-C₈)alkoxy,    (C₁-C₈)alkylthio, mono- or di-(C₁-C₈)alkylamino, (C₃-C₂₀)heteroaryl,    or (C₆-C₂₀)aryl;-   each Z independently represents hydrogen, (C₁-C₈)alkyl, or an    electron-withdrawing group (such as an amide, carboxylic acid,    carboxylic acid ester, halogen, cyano, or nitro), preferably a    hydrogen, (C₁-C₈)alkyl, halogen, cyano, or nitro, most preferably    hydrogen;-   n is an integer from 1 to 3;-   m is 0 or 1, preferably 1; and-   R₁ and R₂ are each independently hydrogen, (C₁-C₈)alkyl, or    (C₃-C₈)cycloalkyl, preferably hydrogen, or R₁ and R₂ taken together    with the carbon atom to which they are attached form a    (C₃-C₈)cycloalkyl ring.

In yet other embodiments, the cleavage group may have a structure of theformula:

-   or a pharmaceutically acceptable salt thereof, wherein-   G represents a sugar, sugar acid, or modified sugar, preferably a    sugar or sugar acid, most preferably glucuronic acid;-   B is a unit covalently attached to the active agent,-   W represents —C(O)—, —C(O)NR′—, —C(O)O—, —S(O)₂NR′—, —P(O)R″NR′—,    —S(O)NR′—, or —PO₂NR′—, in each case where the C(O), S, or P is    directly bound to the phenyl ring, and R′ and R″ are each    independently hydrogen, (C₁-C₈)alkyl, (C₃-C₈)cycloalkyl,    (C₁-C₈)alkoxy, (C₁-C₈)alkylthio, mono- or di-(C₁-C₈)alkylamino,    (C₃-C₂₀)heteroaryl, or (C₆-C₂₀)aryl;-   each Z independently represents hydrogen, (C₁-C₈)alkyl, or an    electron-withdrawing group (such as an amide, carboxylic acid,    carboxylic acid ester, halogen, cyano, or nitro), preferably a    hydrogen, (C₁-C₈)alkyl, halogen, cyano, or nitro, most preferably    hydrogen;-   n is an integer from 1 to 3, preferably 3;-   L represents the a peptide sequence;-   R₁ and R₂ are each independently hydrogen, (C₁-C₈)alkyl, or    (C₃-C₈)cycloalkyl, preferably hydrogen, or R₁ and R₂ taken together    with the carbon atom to which they are attached form a    (C₃-C₈)cycloalkyl ring.

In another aspect, the invention relates to a linker compound, that canbe coupled to an active agent and a ligand (preferably an antibody) toresult in a conjugate of the invention. Accordingly, in such a linkercompound i) the linker comprises a peptide sequence of a plurality ofamino acids; and ii) at least two cleavage groups are covalently coupledto side chains of the amino acids, wherein each cleavage group has areactive moiety capable of reacting with an active agent. The cleavagegroup may have a structure of the formula:

wherein:

B represents a leaving group capable of being displaced an active agent,such as a halogen (esp. Cl or Br), or a unit comprising a reactivemoiety capable of being coupled to an active agent, such as anisocyanate, an acid chloride, a chloroformate, etc.;

G represents a sugar or sugar acid, preferably glucuronic acid;

W represents an electron-withdrawing group, preferably —C(O)NR′—, whereC(O) is bonded to the phenyl ring and NR′ is bonded to L; each Zindependently represents hydrogen, (C₁-C₈)alkyl, or anelectron-withdrawing group (such as an amide, carboxylic acid,carboxylic acid ester, halogen, cyano, or nitro), preferably a hydrogen,(C₁-C₈)alkyl, halogen, cyano, or nitro, most preferably hydrogen;

n is an integer from 1 to 3, preferably 3;

R₁ and R₂ are each independently hydrogen, (C₁-C₈)alkyl, or(C₃-C₈)cycloalkyl, preferably hydrogen, or R₁ and R₂ taken together withthe carbon atom to which they are attached form a (C₃-C₈)cycloalkylring; and

L represents a linkage to the peptide sequence.

Alternatively, the cleavage group may have a structure of the formula:

-   wherein:-   G represents a sugar, sugar acid, or modified sugar, preferably a    sugar or sugar acid, most preferably glucuronic acid;-   W represents —C(O)—, —C(O)NR′—, —C(O)O—, —S(O)₂NR′—, —P(O)R″NR′—,    —S(O)NR′—, or —PO₂NR′—, in each case where the C(O), S, or P is    preferably directly bound to the phenyl ring, and R′ and R″ are each    independently hydrogen, (C₁-C₈)alkyl, mono- or    di-carboxyl(C₁-C₈)alkyl, (C₃-C₈)cycloalkyl, (C₁-C₈)alkoxy,    (C₁-C₈)alkylthio, mono- or di-(C₁-C₈)alkylamino, (C₃-C₂₀)heteroaryl,    or (C₆-C₂₀)aryl;-   each Z independently represents hydrogen, (C₁-C₈)alkyl, or an    electron-withdrawing group (such as an amide, carboxylic acid,    carboxylic acid ester, halogen, cyano, or nitro), preferably a    hydrogen, (C₁-C₈)alkyl, halogen, cyano, or nitro, most preferably    hydrogen;-   n is an integer from 1 to 3;-   m is 0 or 1, preferably 1; and-   R₁ and R₂ are each independently hydrogen, (C₁-C₈)alkyl, or    (C₃-C₈)cycloalkyl, preferably hydrogen, or R₁ and R₂ taken together    with the carbon atom to which they are attached form a    (C₃-C₈)cycloalkyl ring.

Alternatively, the cleavage group may have a structure of the formula:

-   or a pharmaceutically acceptable salt thereof, wherein-   G represents a sugar, sugar acid, or modified sugar, preferably a    sugar or sugar acid, most preferably glucuronic acid;-   B is a unit comprising a reactive moiety capable of being coupled to    the active agent,-   W represents —C(O)—, —C(O)NR′—, —C(O)O—, —S(O)₂NR′—, —P(O)R″NR′—,    —S(O)NR′—, or —PO₂NR′—, in each case where the C(O), S, or P is    directly bound to the phenyl ring, and R′ and R″ are each    independently hydrogen, (C₁-C₈)alkyl, (C₃-C₈)cycloalkyl,    (C₁-C₈)alkoxy, (C₁-C₈)alkylthio, mono- or di-(C₁-C₈)alkylamino,    (C₃-C₂₀)heteroaryl, or (C₆-C₂₀)aryl;-   each Z independently represents hydrogen, (C₁-C₈)alkyl, or an    electron-withdrawing group (such as an amide, carboxylic acid,    carboxylic acid ester, halogen, cyano, or nitro), preferably a    hydrogen, (C₁-C₈)alkyl, halogen, cyano, or nitro, most preferably    hydrogen;-   n is an integer from 1 to 3, preferably 3;-   L represents a linker comprising the peptide sequence;-   R₁ and R₂ are each independently hydrogen, (C₁-C₈)alkyl, or    (C₃-C₈)cycloalkyl, preferably hydrogen, or R₁ and R₂ taken together    with the carbon atom to which they are attached form a    (C₃-C₈)cycloalkyl ring.

In some such embodiments, G is

wherein R₃ is hydrogen or a carboxyl protecting group; andeach R₄ is independently hydrogen or a hydroxyl protecting group.

In some embodiments, the linker comprises a connection unit representedby —(CH₂)_(r)(V(CH₂)_(p))_(q)—, —((CH₂)_(p)V)_(q)—,—(CH₂)_(r)(V(CH₂)_(p))_(q)Y—, —((CH₂)_(p)V)_(q)(CH₂)_(r)—,—Y(((CH₂)_(p)V)_(q)— or —(CH₂)_(r)(V(CH₂)_(q)YCH₂—

wherein:

-   -   r is an integer from 0 to 10;    -   p is an integer from 1 to 10;    -   q is an integer from 1 to 20;    -   V and Y are each independently a single bond, —O—, —S—, —NR₂₁—,        —C(O)NR₂₂—, —NR₂₃C(O)—, —NR₂₄SO₂—, or —SO₂NR₂₅—; and    -   R₂₁ to R₂₅ are each independently hydrogen, (C₁-C₆)alkyl,        (C₁-C₆)alkyl(C₆-C₂₀)aryl or (C₁-C₆)alkyl(C₃-C₂₀)heteroaryl.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 illustrates an active drug release mechanism from a β-glucuronidebased linker.

FIG. 2 is a graph depicting the hydrolysis of a linker byβ-glucuronidase from Experimental Example 1.

FIG. 3 is a graph depicting the plasma stability of two drug-linkerconjugates from Experimental Example 2.

FIG. 4 is a graph depicting the plasma stability of compound 47a,described in Example 68.

FIG. 5 is a graph depicting the plasma stability of compound 49a,described in Example 70.

FIG. 6 is a graph depicting the plasma stability of compound 48a,described in Example 69.

FIG. 7 consists of two panels, FIG. 7A and FIG. 7B. FIG. 7A displays astrategy for conjugating a drug to an antibody (DAR2). FIG. 7B displaysa strategy for conjugating a drug to an antibody (DAR4).

FIG. 8 shows relative in vitro activities of the DAR2 MMAE-conjugates,with varying PEG length in the linker, against JIMT-1 (HER2 positive)and MCF7 cells (HER2 negative).

FIG. 9 shows relative in vitro activities of the DAR4 MMAE-conjugates,which have a different PEG length in linker part, against JIMT-1 (HER2positive) and MCF7 cells (HER2 negative).

FIG. 10 shows human beta-glucuronidase reactivity in DAR4 ADCs, whichhave the various linker types. 12 μM of ADCs were incubated with 0.01 μgof human beta-glucuronidase (R&D Systems) for 3 hours at 37° C.

FIG. 11 shows plasma stability of ADC2 and Kadcyla in mouse or humanplasma.

FIG. 12 shows human plasma stability of ADC33 and ADC34 for 7 days.

FIG. 13 shows Rat PK profile of Herceptin and ADC2.

FIG. 14 shows Rat PK profile of ADC23 and ADC34.

FIG. 15 shows Rat PK profile improvement of MMAE-based ADCs by replacinglinker-toxin from 2g to 11j.

FIG. 16 shows Rat PK profile improvement by branched linker unit.

FIG. 17 shows impact of polar amino acid on Rat PK profile in MMAE ADC.

FIG. 18 shows Rat PK profile improvement by branched linker-toxin withor without polar amino acid in ADC with DAR2.

FIG. 19 shows effects of Asp in linker-toxin unit on Rat PK profile ofADC with DAR4.

FIG. 20 shows effects of Glu in linker-toxin unit on Rat PK profile ofADC with DAR4.

FIG. 21 shows in vivo efficacy of representative amine type DAR4 ADCusing MMAF (ADC23) or MMAE (ADC24).

FIG. 22 shows in vivo efficacy of representative amide type DAR4 ADCusing MMAF (ADC34) or MMAE (ADC33).

DETAILED DESCRIPTION

The present invention relates to antibody-drug conjugates (ADCs) whereina plurality of drugs are conjugated to an antibody through a linker. Thelinker preferably comprises a peptide sequence of a plurality of aminoacids, wherein at least two of the active agents are covalently coupledto side chains of the amino acids. However, as one of skill in the artwould recognize, the antibody portion of such conjugates can be replacedby any suitable ligand, and thus the invention relates in equal measureto ligand-drug conjugates. Accordingly, references to and discussions ofantibody-drug conjugates herein should be understood, where notcontradicted by context, as equally applicable to ligand-drug conjugatesand their corresponding intermediates (e.g., ligand-linker conjugates).In all aspects related to the various ligand-drug conjugates disclosedherein, however, the ligand is preferably an antibody.

In certain such embodiments, two or more such linkers are conjugated tothe antibody, e.g., 2-4 linkers, which may each be coupled to adifferent C-terminal cysteine of a heavy or light chain of the antibody.

An active agent may be covalently coupled to the side chain of an aminoacid, termed a “linking amino acid” herein. In certain preferredembodiments, a linking amino acid, such as each linking amino acid in alinker, is lysine or ornithine. Nevertheless, many other amino acids maybe linking amino acids in various embodiments of the invention. Forexample, a linking amino acid may be selected from lysine,5-hydroxylysine, 4-oxalysine, 4-thialy sine, 4-selenalysine,4-thiahomolysine, 5,5-dimethyllysine, 5,5-difluorolysine,trans-4-dehydrolysine, 2,6-diamino-4-hexynoic acid, cis-4-dehydrolysine,6-N-methyllysine, diaminopimelic acid, ornithine, 3-methylornithine,α-methylornithine, citrulline, and homocitrulline. A linking amino acidmay be a L-amino acid or a D-amino acid. A linking amino acid may be anα-amino acid or a β-amino acid. A linking amino acid may be anaturally-occurring amino acid or a non-naturally-occurring amino acid.

The peptide may comprise 2 to 20 amino acids. The majority of aminoacids of the peptide may be independently selected from alanine,aspartate, asparagine, glutamate, glutamine, glycine, lysine, ornithine,proline, serine, and threonine. For example, each amino acid of thepeptide may be independently selected from alanine, aspartate,asparagine, glutamate, glutamine, glycine, lysine, ornithine, proline,serine, and threonine.

In preferred embodiments, the peptide comprises at least one hydrophilicamino acid, e.g., in addition to the linking amino acids. Hydrophilicamino acids may increase the water solubility of the antibody-drugconjugate, linker, and/or precursors of the antibody-drug conjugate.Each hydrophilic amino acid may be a naturally-occurring amino acid or anon-naturally-occurring amino acid. A hydrophilic amino acid may be anα-amino acid or a β-amino acid. A hydrophilic amino acid may bearginine, aspartate, asparagine, glutamate, glutamine, histidine,lysine, ornithine, proline, serine, or threonine, and may be a D-aminoacid or an L-amino acid. In preferred embodiments, the peptide comprisesa hydrophilic amino acid selected from aspartate or glutamate, such asL-aspartate or L-glutamate. In other preferred embodiments, the peptidecomprises a hydrophilic amino acid selected from lysine or arginine,such as L-lysine or L-arginine. In certain embodiments, the peptidecomprises a hydrophilic amino acid with a side chain having a moietythat bears a charge at neutral pH in aqueous solution (e.g., an amine,guanidine, or carboxyl moiety).

The peptide may comprise naturally-occurring amino acids and/ornon-naturally-occurring amino acids. The peptide may comprise α-aminoacids and/or β-amino acids. In some embodiments, the peptide consistsessentially of α-amino acids. In some embodiments, the peptide consistsessentially of naturally-occurring amino acids (i.e., wherein thelinking amino acids are naturally-occurring amino acids, such as lysineor ornithine, that are substituted with a group comprising an activeagent). The peptide may comprise, consist essentially of, or evenconsist of amino acids selected from alanine, aspartate, asparagine,glutamate, glutamine, glycine, lysine, ornithine, proline, serine, andthreonine, any of which may be L-amino acids and/or D-amino acids. Insome embodiments, the peptide consists essentially of L-amino acids. Incertain embodiments, the peptide does not comprise a hydrophobic aminoacid, such as an amino acid selected from isoleucine, methionine,leucine, phenylalanine, tryptophan, tyrosine, or valine; in other words,in such embodiments, the peptide is free or essentially free of theseamino acids. In preferred embodiments, the peptide does not comprise anyone of isoleucine, methionine, leucine, phenylalanine, tryptophan,tyrosine, and valine.

In addition to the peptide, the linker may comprise an O-substitutedoxime. In preferred embodiments, the oxygen atom of the oxime issubstituted with a group that covalently links the oxime to the peptide,and the carbon atom of the oxime is substituted with a group thatcovalently links the oxime to the antibody. In other embodiments, thecarbon atom of the oxime is substituted with a group that covalentlylinks the oxime to the peptide, and the oxygen atom of the oxime issubstituted with a group that covalently links the oxime to theantibody. The oxime may covalently link the antibody to the N-terminusof the peptide or the C-terminus of the peptide, or the oxime maycovalently link the antibody to a side chain of the peptide. In someembodiments, the linker does not comprise an oxime. For example, thelinker may comprise a product of a cycloaddition reaction, such as asubstituted triazole, instead of an oxime.

Active agents may be coupled to a linker by cleavable or non-cleavablebonds, hydrolysable or non-hydrolyzable bonds. In certain preferredembodiments, the active agents are coupled to the linker by cleavablebonds. For example, an antibody-drug conjugate may comprise aself-immolative group, preferably a self-immolative group for eachactive agent, e.g., to release an active agent from the ADC.

For example, one or more active agents, or even each active agent, maybe coupled to the linker through a cleavage group of Formula (I)

-   wherein-   G is a sugar or sugar acid, preferably glucuronic acid or a    derivative thereof;-   B represents the active agent, such as a drug;-   W represents an electron-withdrawing group, preferably —C(O)NR′—,    where C(O) is bonded to the phenyl ring and NR′ is bonded to L;-   each Z independently represents hydrogen, (C₁-C₈)alkyl, or an    electron-withdrawing group (such as an amide, carboxylic acid,    carboxylic acid ester, halogen, cyano, or nitro), preferably a    hydrogen, (C₁-C₈)alkyl, halogen, cyano, or nitro, most preferably    hydrogen;-   n is an integer from 0 to 3, preferably 3;-   L comprises a chain of 20 to 100 atoms, comprising at least one    amino acid of the peptide, that covalently links the antibody to W;    and-   R₁ and R₂ are each independently hydrogen, (C₁-C₈)alkyl, or    (C₃-C₈)cycloalkyl, preferably hydrogen, or R₁ and R₂ taken together    with the carbon atom to which they are attached form a    (C₃-C₈)cycloalkyl ring.

Alternatively, the cleavage group may have a structure of the formula:

-   wherein:-   G represents a sugar, sugar acid, or modified sugar, preferably a    sugar or sugar acid, most preferably glucuronic acid;-   W represents —C(O)—, —C(O)NR′—, —C(O)O—, —S(O)₂NR′—, —P(O)R″NR′—,    —S(O)NR′—, or —PO₂NR′—, in each case where the C(O), S, or P is    preferably directly bound to the phenyl ring, and R′ and R″ are each    independently hydrogen, (C₁-C₈)alkyl, mono- or    di-carboxyl(C₁-C₈)alkyl, (C₃-C₈)cycloalkyl, (C₁-C₈)alkoxy,    (C₁-C₈)alkylthio, mono- or di-(C₁-C₈)alkylamino, (C₃-C₂₀)heteroaryl,    or (C₆-C₂₀)aryl;-   each Z independently represents hydrogen, (C₁-C₈)alkyl, or an    electron-withdrawing group (such as an amide, carboxylic acid,    carboxylic acid ester, halogen, cyano, or nitro), preferably a    hydrogen, (C₁-C₈)alkyl, halogen, cyano, or nitro, most preferably    hydrogen;-   n is an integer from 1 to 3;-   m is 0 or 1, preferably 1; and-   R₁ and R₂ are each independently hydrogen, (C₁-C₈)alkyl, or    (C₃-C₈)cycloalkyl, preferably hydrogen, or R₁ and R₂ taken together    with the carbon atom to which they are attached form a    (C₃-C₈)cycloalkyl ring.

Alternatively, the cleavage group may have a structure of the formula:

-   or a pharmaceutically acceptable salt thereof, wherein-   G represents a sugar, sugar acid, or modified sugar, preferably a    sugar or sugar acid, most preferably glucuronic acid;-   B is a unit comprising a reactive moiety capable of being coupled to    the active agent,-   W represents —C(O)—, —C(O)NR′—, —C(O)O—, —S(O)₂NR′—, —P(O)R″NR′—,    —S(O)NR′—, or —PO₂NR′—, in each case where the C(O), S, or P is    directly bound to the phenyl ring, and R′ and R″ are each    independently hydrogen, (C₁-C₈)alkyl, (C₃-C₈)cycloalkyl,    (C₁-C₈)alkoxy, (C₁-C₈)alkylthio, mono- or di-(C₁-C₈)alkylamino,    (C₃-C₂₀)heteroaryl, or (C₆-C₂₀)aryl;-   each Z independently represents hydrogen, (C₁-C₈)alkyl, or an    electron-withdrawing group (such as an amide, carboxylic acid,    carboxylic acid ester, halogen, cyano, or nitro), preferably a    hydrogen, (C₁-C₈)alkyl, halogen, cyano, or nitro, most preferably    hydrogen;-   n is an integer from 1 to 3, preferably 3;-   L represents a linker comprising the peptide sequence;-   R₁ and R₂ are each independently hydrogen, (C₁-C₈)alkyl, or    (C₃-C₈)cycloalkyl, preferably hydrogen, or R₁ and R₂ taken together    with the carbon atom to which they are attached form a    (C₃-C₈)cycloalkyl ring.

The electron withdrawing group W may be —C(O)—, —C(O)NR′—, —C(O)O—,—SO₂NR′—, —P(O)R″NR′—, —SONR′—, or —PO₂NR′—, preferably —C(O)NR′—, andR′ and R″ may be each independently hydrogen, (C₁-C₈)alkyl,(C₃-C₈)cycloalkyl, (C₁-C₈)alkoxy, (C₁-C₈)alkylthio, mono- ordi-(C₁-C₈)alkylamino, (C₃-C₂₀)heteroaryl, or (C₆-C₂₀)aryl, preferablyhydrogen. In such embodiments, W is preferably oriented such that thecarbonyl, phosphoryl, sulphonyl, or sulphinyl group is directly bound tothe phenyl ring. Where Z represents an electron-withdrawing group, Z mayrepresent any of the moieties described in this paragraph for W.

W may represent —C(O)NR′—, and the nitrogen of W may be a nitrogen atomof the side chain of a linking amino acid, such as lysine or ornithine.Similarly, W may represent —C(O)NR′—, and the nitrogen of W may be anitrogen atom of the N-terminal amino acid in the peptide, e.g., theamine nitrogen (backbone nitrogen) of the N-terminal amino acid.

The sugar or sugar acid of the cleavage group (i.e., G) is preferablylinked to the phenyl ring, e.g., by a bond susceptible to enzymaticcleavage, such as a glycosidic bond to an oxygen substituent on thephenyl ring. The sugar or sugar acid is preferably a monosaccharide,such as glucuronic acid or a derivative thereof, which is capable ofbeing cleaved from the ADC by an enzyme, such as a β-glucuronidase,e.g., an enzyme present in cells to be targeted by the conjugate.Glucuronic acid and derivatives thereof may be represented by Formula(II):

wherein R₃ is hydrogen or a carboxyl protecting group, preferablyhydrogen, and each R₄ is independently hydrogen or a hydroxyl protectinggroup, preferably hydrogen.

A carboxyl protecting group may be any suitable protecting group formasking a carboxylic acid, e.g., in organic synthesis, such as methyl,methoxymethyl, methylthiomethyl, tetrahydropyranyl, benzyloxymethyl,phenacyl, N-phthalimidomethyl, 2,2,2-trichloroethyl, 2-haloethyl,2-(p-toluenesulfonyl)ethyl, t-butyl, cinnamyl, benzyl, triphenylmethyl,bis(o-nitrophenyl)methyl, 9-anthrylmethyl, 2-(9,10-dioxo)anthrylmethyl,piperonyl, 2-trimethylsilylethyl, trimethylsilyl, ort-butyldimethylsilyl. In some embodiments, the entire moiety R₃—OC(═O)—is replaced by a carboxyl-masking moiety such as 2-alkyl-1,3-oxazolinyl.

A hydroxyl protecting group may be any suitable protecting groupsuitable for masking a hydroxyl group, e.g., in organic synthesis, suchas acetyl, methyl, ethoxyethyl, benzoyl, benzyl, 4-methoxybenzyl,3,4-dimethoxybenzyl, tetrahydropyranyl (THP), tetrahydrofuranyl (THF),tert-butyldimethylsilyl (TBDMS), trimethylsilyl (TMS), triethylsilyl(TES), triisopropylsilyl (TIPS), tert-butyldiphenylsilyl (TBDPS),tri-isopropylsilyloxymethyl (TOM), β-methoxyethoxymethyl (MEM),methoxymethyl (MOM), allyl, or trityl.

L and/or the linker may comprise a substituted or unsubstituted alkylenehaving 1 to 100 carbon atoms, preferably 20 to 80 carbon atoms, andsatisfy at least one, preferably at least two, of the following (i) to(iv):

-   -   (i) the alkylene includes at least one unsaturated bond,        preferably 3 or 4 double bonds and no triple bonds,    -   (ii) the alkylene includes at least one heteroarylene,    -   (iii) at least one carbon atom of the alkylene is replaced by        one or more heteroatoms selected from nitrogen (N), oxygen (O),        and sulfur (S), preferably at least one nitrogen and at least        one oxygen (e.g., as in an oxime), and    -   (iv) the alkylene is substituted with one or more alkyls having        1 to 20 carbon atoms, preferably 2 or 3 methyls.

In preferred embodiments, a cysteine of the antibody, preferably at aC-terminus of a heavy or light chain of the antibody, forms a thioetherbond with a carbon atom of an isoprenyl unit, thereby covalently linkingthe antibody to the linker. Thus, in some embodiments, L and/or thelinker may comprise at least one isoprenyl unit, preferably twoisoprenyl units, each represented by Formula (III), which is preferablyrecognizable by an isoprenoid transferase, e.g., as part of a product orsubstrate of the isoprenoid transferase.

In certain such preferred embodiments, the antibody comprises an aminoacid motif capable of being recognized by an isoprenoid transferase. Forexample, at least one C-terminus of the antibody may comprise an aminoacid motif capable of being recognized by an isoprenoid transferase(e.g., as a substrate, for example, prior to forming the antibody-drugconjugate, or as a product of an isoprenoid transferase, for example,after forming the antibody-drug conjugate). The antibody may furthercomprise a spacer, such as an amino acid or a stretch of amino acidsthat links a peptide chain of the antibody to the amino acid motif. Thespacer may consist of 1 to 20 consecutive amino acids, preferably 7 ormore amino acids. In some embodiments, glycine and proline are preferredamino acids for the spacer, and may be used in any combination, such asa series of about 7 glycines. In other embodiments, the amino acids ofthe spacer are each independently selected from glycine, aspartic acid,arginine, and serine. The antibody may comprise an addition or deletionat a carboxy terminus, e.g., relative to a form of the antibody notincluded in an ADC.

Examples of isoprenoid transferases include farnesyl protein transferase(FTase) and geranylgeranyl transferase (GGTase), which can catalyze thetransfer of a farnesyl or geranyl-geranyl group to at least oneC-terminal cysteine of a target protein. A GGTase may be classified aseither GGTase I or GGTase II. FTase and GGTase I may recognize a CAAXmotif, and GGTase II may recognize a XXCC, XCXC, or CXX motif, wherein Crepresents cysteine, A represents an aliphatic amino acid (e.g.,isoleucine, valine, methionine, leucine), and each X independentlyrepresents, for example, glutamine, glutamate, serine, cysteine,methionine, alanine, or leucine (see Nature Rev. Cancer, 5(5):405-12(2005); Nature Chemical Biology 17:498-506 (2010); Lane K T, Bees L S,J. Lipid Research, 47:681-699 (2006); Kasey P J, Seabra M C, J.Biological Chemistry, 271(10):5289-5292 (1996), each of which is herebyincorporated by reference in its entirety).

The antibody-drug conjugates according to the present invention maycomprise an amino acid motif, such as CYYX, XXCC, XCXC, or CXX,preferably CYYX (wherein, C represents cysteine, Y represents analiphatic amino acid, such as leucine, isoleucine, valine, and/ormethionine, and X represents an amino acid that determines a substratespecificity of the isoprenoid transferase, such as glutamine, glutamate,serine, cysteine, methionine, alanine, and/or leucine).

Isoprenoid transferases from various sources may be used. For example,the isoprenoid transferase may be obtained from a human, animal, plant,bacteria, virus, or other source. In some embodiments, a naturallyoccurring isoprenoid transferase is used. In some embodiments, anaturally-modified or artificially-modified isoprenoid transferase maybe used. For example, the isoprenoid transferase may comprise one ormore amino acid substitutions, additions, and/or deletions, and/or theisoprenoid transferase may be modified by the addition of at least oneof Histidine-tag, GST, GFP, MBP, CBP, Isopeptag, BCCP, Myc-tag,Calmodulin-tag, FLAG-tag, HA-tag, Maltose binding protein-tag, Nus-tag,Glutathione-S-transferase-tag, Green fluorescent protein-tag,Thioredoxin-tag, S-tag, Softag 1, Softag 3, Strep-tag, SBP-tag, Ty-tag,and the like.

Isoprenoid transferases recognize an isosubstrate and/or a substrate.The term isosubstrate refers to a substrate analog comprising a chemicalmodification. Isoprenoid transferases can alkylate a specific amino acidmotif (for example, a CAAX motif) at the C-terminus of an antibody (see,e.g., Duckworth, B P et al., ChemBioChem, 8:98 (2007); Uyen T T et al.,ChemBioChem, 8:408 (2007); Labadie, G R et al., J. Org. Chem.,72(24):9291 (2007); Wollack, J W et al., ChemBioChem, 10:2934 (2009),each of which is hereby incorporated by reference). A functionalizedantibody may be produced using an isoprenoid transferase and anisosubstrate, which may alkylate a C-terminal cysteine.

The isosubstrate may be, for example, the compound of Formula IV.

The cysteine of a C-terminal CAAX motif may be bound to an isosubstrateusing an isoprenoid transferase. In some embodiments, part of the motif,e.g., AAX, may subsequently be removed by a protease, e.g., leaving onlythe cysteine to which the isoprenoid is bound. The cysteine mayoptionally be methylated at the carboxyl terminus, e.g., by an enzyme(see, e.g., Bell, I M, J. Med. Chem., 47(8):1869 (2004), which is herebyincorporated by reference).

L and/or the linker may comprise a binding unit formed by a 1,3-dipolarcycloaddition reaction, hetero-Diels-Alder reaction, nucleophilicsubstitution reaction, non-aldol type carbonyl reaction, addition to acarbon-carbon multiple bond, oxidation reaction, or click reaction. Abinding unit may be formed by a reaction between an acetylene and azide,or a non-aldol type carbonyl reaction, such as a reaction between analdehyde or ketone group and hydrazine or alkoxyamine; such bindingunits may be represented by Formula (A), (B), (C), or (D).

L₁ is a single bond or alkylene having 1 to 30 carbon atoms, preferably10, 11, 12, 13, 14, 15, or 16 carbon atoms;R₁₁ is hydrogen or an alkyl having 1 to 10 carbon atoms, preferablymethyl; andL₂ is an alkylene having 1 to 30 carbon atoms, preferably 10, 11, 12,13, 14, 15, or 16 carbon atoms.

In some embodiments, L₁ and/or L₂ may comprise at least one isoprenylunit, represented by Formula (III), preferably two isoprenyl units. L₂may consist of at least one isoprenyl unit, represented by Formula(III), preferably two isoprenyl units. In preferred embodiments, acarbon atom of an isoprenyl unit forms a thioether bond with the sulfuratom of a cysteine of the antibody, most preferably at a C-terminus of aheavy or light chain, thereby covalently linking the antibody and thelinker.

An antibody-drug conjugate may comprise the binding unit represented byFormula (D) supra, wherein L₂ consists of at least one isoprenyl unit,preferably two isoprenyl units. The binding unit may be an O-substitutedoxime, i.e., the nitrogen of the binding unit may be covalently bound toa substituted oxygen. A carbon atom of an isoprenyl unit may form athioether bond with the sulfur atom of a cysteine of the antibody, mostpreferably at a C-terminus of a heavy or light chain, thereby covalentlylinking the binding unit and the antibody.

L and/or the linker may comprise an isoprenyl group represented by

e.g., wherein a carbon atom of the isoprenyl group forms a thioetherbond with a sulfur atom of a cysteine of the antibody, therebycovalently linking the isoprenyl group and the antibody. The nitrogen ofthe isoprenyl group may covalently link the isoprenyl group to apolyethylene glycol unit of L and/or the linker.

In some embodiments, L and/or the linker may comprise an isoprenyl grouprepresented by e.g.,

wherein a carbon atom of the isoprenyl group forms a thioether bond witha sulfur atom of a cysteine of the antibody, thereby covalently linkingthe isoprenyl group and the antibody. The nitrogen of the isoprenylgroup may covalently link the isoprenyl group to a polyethylene glycolunit of L and/or the linker.

Click chemistry reactions may be carried out under mild conditions,which can be performed in the presence of an antibody without denaturingthe antibody. A click chemistry reaction shows high reactionspecificity. Therefore, even though antibodies have various functionalgroups (for example, amines, carboxyls, carboxamides, and guanidiniums),a click chemistry reaction may be performed, for example, withoutaffecting the amino acid side chains of the antibody. A click chemistryreaction between an azide group and an acetylene group, for example, mayoccur in the presence of an antibody without modifying the amino acidside chain functional groups of the antibody. Further, a click chemistryreaction may precisely target a specific functional group, such asfunctional groups rarely found in nature, regardless of the nature ofthe reactants. In some cases, the reactants are selected to improveoverall reaction efficiency. For example, an azide-acetylene clickchemistry reaction may produce triazole with a high yield (see, e.g.,Hia, R K et al., Chem. Rev., 109:5620 (2009); Meldal, M & Tornoe, C W,Chem Rev., 108:2952 (2008); Kolb, H C et al., Angew. Chemie Int. Ed.Engl., 40:2004 (2001), each of which is hereby incorporated byreference).

Azide and acetylene functional groups do not exist in natural proteins.Thus, none of the amino acid side chains, N-terminal amines, orC-terminal carboxyls should be affected by a click chemistry reactionthat utilizes these functional groups.

The L moiety of Formula I and/or the linker may further include aconnection unit represented by —(CH₂)_(r)(V(CH₂)_(p))_(q)— or—(CH₂CH₂X)_(w)—, wherein

V is a single bond, —O—, —S—, —NR₂₁—, —C(O)NR₂₂—, —NR₂₃C(O)—, —NR₂₄SO₂—,or —SO₂NR₂₅—, preferably —O—;

X is —O—, (C₁-C₈)alkylene, or —NR₂₁—, preferably —O—;

R₂₁ to R₂₅ are each independently hydrogen, (C₁-C₆)alkyl,(C₁-C₆)alkyl(C₆-C₂₀)aryl, or (C₁-C₆)alkyl(C₃-C₂₀)heteroaryl, preferablyhydrogen;

r is an integer from 1 to 10, preferably 2 or 3;

p is an integer from 0 to 12, preferably 1 or 2;

q is an integer from 1 to 20, preferably 4 to 20; and

w is an integer from 1 to 20, preferably 4 to 20.

L and/or the linker preferably comprise the binding unit represented byFormula (A), (B), (C), or (D) and the connection unit represented by—(CH₂)_(r)(V(CH₂)_(p))_(q)— or —(CH₂CH₂X)_(w)—.

In preferred embodiments, L and/or the linker comprise at least onepolyethylene glycol unit represented by either

The antibody-drug conjugate may comprise from 1 to 20 —OCH₂CH₂— units,such as 1 to 12 —OCH₂CH₂— units, 5 to 12 —OCH₂CH₂— units, 6 to 12—OCH₂CH₂— units, 5 to 20 —OCH₂CH₂— units, or 6 to 20 —OCH₂CH₂— units. Inembodiments wherein L and/or the linker comprises an oxime, apolyethylene glycol unit preferentially covalently links the oxime tothe peptide, e.g., the N-terminus of the peptide, the C-terminus of thepeptide, or a side chain of the peptide.

L and/or the linker preferably comprises a polyethylene glycol grouprepresented by —(CH₂CH₂O)_(n)—, wherein n is 1 to 20, such as 1 to 12, 5to 12, 6 to 12, 5 to 20, or 6 to 20. In embodiments wherein L and/or thelinker comprises an oxime, a polyethylene glycol group preferentiallycovalently links the oxime to the peptide.

In some embodiments, L and/or the linker preferably comprises one of thefollowing two structures:

wherein n is an integer from 1 to 20, such as 4 to 20.

-   L and/or the linker may comprise

wherein

-   V represents a single bond, —O—, —S—, —NR₂₁—, —C(O)NR₂₂—,    —NR₂₃C(O)—, —NR₂₄SO₂—, or —SO₂NR₂₅—, preferably —O—;-   R₂₁ to R₂₅ represents each independently hydrogen, (C₁-C₆)alkyl,    (C₁-C₆)alkyl(C₆-C₂₀)aryl, or (C₁-C₆)alkyl(C₃-C₂₀)heteroaryl;-   r is an integer from 1 to 10, preferably 2 or 3;-   p is an integer from 0 to 10, preferably 1 or 2;-   q is an integer from 1 to 20, preferably 1 to 6; and-   L₁ is a single bond.

In some embodiments, at least two active agents are covalently coupledto side chains of the amino acids through one or more linkers,optionally including a cleavage group, as disclosed herein, e.g., thatis capable of releasing the active agent from the conjugate in a desiredlocation. Any or all of the linkers may be a branched linker, e.g.,wherein each branch of the linker terminates in an active agent (e.g.,linked through a cleavage group), or wherein one or more branches of thelinker terminate in an active agent (e.g., linked through a cleavagegroup) and one or more others comprise a polyethylene glycol moiety anddo not include an active agent. In some such embodiments, each branchedlinker is coupled to at least two active agents, which may be the sameor different. In some embodiments, at least two, three, or four branchedlinkers are covalently coupled to the peptide. In other embodiments,exactly one branched linker is coupled to the peptide.

A branched linker may comprise a branching unit, such that each activeagent is coupled to the branching unit through a secondary linker andthe branching unit is coupled to the peptide by a primary linker.

A branching unit can have any suitable structure, such as:

-   wherein L₁, L₂, L₃ is each independently a direct bond or    —C_(n)H_(2n)— where n is a integer of 1 to 30,-   wherein G₁, G₂, G₃ is each independently a direct bond,

wherein R₃ is hydrogen or C₁-C₃₀ alkyl;

-   wherein R₄ is hydrogen or -L₄-COOR₅, wherein L₄ is a direct bond or    —C_(n)H_(2n)— wherein n is a integer of 1 to 10, and R₅ is hydrogen    or C₁-C₃₀ alkyl.

A secondary linker (e.g., linking an active agent to the peptide) maycomprise a binding unit formed by a 1,3-dipolar cycloaddition reaction,hetero-Diels-Alder reaction, nucleophilic substitution reaction,non-aldol type carbonyl reaction, addition to a carbon-carbon multiplebond, oxidation reaction, or click reaction. A binding unit may beformed by a reaction between an acetylene and azide, or a non-aldol typecarbonyl reaction, such as a reaction between an aldehyde or ketonegroup and hydrazine or alkoxyamine, reactions which allow for mildcoupling of active agents and/or cleavage groups to the peptidesequence. Such binding units may be represented by Formula (A), (B),(C), or (D).

L₁ is a single bond or alkylene having 1 to 30 carbon atoms, preferably10, 11, 12, 13, 14, 15, or 16 carbon atoms;R₁₁ is hydrogen or an alkyl having 1 to 10 carbon atoms, preferablymethyl; andL₂ is an alkylene having 1 to 30 carbon atoms, preferably 10, 11, 12,13, 14, 15, or 16 carbon atoms.

In some embodiments, the antibody-drug conjugate comprises the structureof Formula (V):

wherein:

A represents the antibody;

P represents the peptide;

B₁ represents a first active agent;

B₂ represents a second active agent; and

n is an integer from 1 to 20.

In some embodiments, the antibody-drug conjugate comprises the structureof Formula (VI):

wherein:

A represents the antibody;

P represents the peptide;

B₁ represents a first active agent;

B₂ represents a second active agent; and

n is an integer from 1 to 20.

In some embodiments, the conjugate comprises a structure of the formula:

where

A represents the ligand;

P

P represents the peptide;

B₁ represents a first active agent;

B₂ represents a second active agent;

L₁ represents a linker, optionally including a cleavage group;

L₂ represents a linker, optionally including a cleavage group; and

n is an integer from 1 to 20.

In some embodiments, the conjugate comprises a structure of the formula:

wherein

A represents the ligand;

P

P represents the peptide;

B₁ represents a first active agent;

B₂ represents a second active agent;

B₃ represents a third active agent;

L₁ represents a linker, optionally including a cleavage group;

L₂ represents a linker, optionally including a cleavage group;

L₃ represents a linker, optionally including a cleavage group; and

n is an integer from 1 to 20.

In some embodiments, the conjugate comprises a structure of the formula:

wherein

A represents the ligand;

P

P represents the peptide;

B₁ represents a first active agent;

B₁₁ represents a second active agent;

B₁₂ represents a third active agent;

L₁ represents a linker;

L₂ represents a linker;

L₁₁ represents a secondary linker, optionally including a cleavagegroup;

L₁₂ represents a secondary linker, optionally including a cleavagegroup;

BU represents a branching unit; and

n is an integer from 1 to 20.

In some embodiments, the conjugate comprises a structure of the formula:

wherein

A represents the ligand;

P

P represents the peptide;

B₁ represents a first active agent;

B₂ represents a second active agent;

L₁ represents a linker, optionally including a cleavage group;

L₂ represents a linker, optionally including a cleavage group; and

n is an integer from 1 to 20.

In some embodiments, the conjugate comprises a structure of the formula:

wherein

A represents the ligand;

P

P represents the peptide;

B₁ represents a first active agent;

B₂ represents a second active agent;

B₃ represents a third active agent;

L₁ represents a linker, optionally including a cleavage group;

L₂ represents a linker, optionally including a cleavage group;

L₃ represents a linker, optionally including a cleavage group; and

n is an integer from 1 to 20.

In some embodiments, the conjugate comprises a structure of the formula:

wherein

A represents the ligand;

P

P represents the peptide;

B₁ represents a first active agent;

B₁₁ represents a second active agent;

B₁₂ represents a third active agent;

L₁ represents a linker;

L₂ represents a linker;

L₁₁ represents a secondary linker, optionally including a cleavagegroup;

L₁₂ represents a secondary linker, optionally including a cleavagegroup;

BU represents a branching unit; and

n is an integer from 1 to 20.

In some embodiments, the antibody-drug conjugate has a structure ofFormula (VII) or a pharmaceutically acceptable salt thereof:

wherein B represents an active agent; mAb represents the antibody; Rrepresents an amino acid side chain; J represents a proton, an acylgroup, or a group comprising an active agent; m is an integer from 0 to10; n is an integer from 0 to 10; p is an integer from 0 to 10; q is aninteger from 1 to 20, preferably 4 to 20; and r is an integer from 0 to10, preferably 1. Each B may be the same active agent or each B may be adifferent active agent. Similarly, each R may be the same amino acidside chain or each R may be a different amino acid side chain. One ormore R's may covalently link one or more additional active agents to thepeptide. Each R group may be the R group of an L-amino acid or a D-aminoacid. Formula (VII) depicts that each amino acid of the peptide is anα-amino acid. Nevertheless, the peptide may comprise one or more β-aminoacids. The structure shows that each linking amino acid is anN-substituted lysine. Nevertheless, an antibody-drug conjugate maycomprise other linking amino acids instead of or in addition to lysine.In some embodiments, J comprises a moiety with a structure of Formula(VIII):

wherein K may be a single bond that links the structure of Formula(VIII) to the N-terminal amide of the peptide, or K is—N(H)(CH₂CH₂O)_(k)—, wherein k is an integer from 1 to 20, preferablyfrom 1 to 10.

In some embodiments, the antibody-drug conjugate has a structure ofFormula (IX):

wherein B represents an active agent; mAb represents the antibody; Rrepresents an amino acid side chain; J represents a hydroxyl, an amidegroup, an amine, or a group comprising an active agent; q is an integerfrom 0 to 10, preferably 1; r is an integer from 0 to 20, preferably3-20; s is an integer from 0 to 10; t is an integer from 0 to 10; and vis an integer from 0 to 10. Each B may be the same active agent or eachB may be a different active agent. Similarly, each R may be the sameamino acid side chain or each R may be a different amino acid sidechain. One or more R's may covalently link one or more additional activeagents to the peptide. Each R may be the R group of an L-amino acid or aD-amino acid. Formula (IX) depicts that each amino acid of the peptideis an α-amino acid. Nevertheless, the peptide may comprise one or moreβ-amino acids. The structure shows that each linking amino acid is anN-substituted lysine. Nevertheless, an antibody-drug conjugate maycomprise other linking amino acids instead of or in addition to lysine.In some embodiments, J comprises a moiety with a structure of Formula(VIII):

wherein K may be —N(H)(CH₂CH₂O)_(k)(CH₂)_(m)N(H)—, wherein k is aninteger from 1 to 20 and m is an integer from 0 to 5.

The antibody-drug conjugates of the invention may be prepared using anymethod known in the art, including molecular biology and cell biologymethods. For example, transient or stable transfection methods may beused. Genetic sequences encoding a specific amino acid motif capable ofbeing recognized by an isoprenoid transferase may be inserted into aknown plasmid vector using standard PCR and/or ligation technologies soas to express an antibody having the specific amino acid motif at aC-terminus thereof. An antibody having at least one amino acid motifcapable of being recognized by the isoprenoid transferase may thus beexpressed in a suitable host, e.g., a CHO cell or in E coli.

The term “antibody” refers to an immunoglobulin molecule that recognizesand specifically binds to a different molecule through at least oneantigen recognition site within a variable region of the immunoglobulinmolecule. As used herein, the term “antibody” includes intact polyclonalantibodies, intact monoclonal antibodies, antibody fragments (forexample, Fab, Fab′, F(ab′)₂, Fd, and Fv fragments), single chain Fv(scFv) mutants, multispecific antibodies such as bispecific antibodiesgenerated from two or more intact antibodies, chimeric antibodies,humanized antibodies, human antibodies, fusion proteins including anantigen determination portion of an antibody, and any other modifiedimmunoglobulin molecule including an antigen recognition site. Theantibody may be any of the five major classes of immunoglobulins: IgA,IgD, IgE, IgG, and IgM, or subclasses (isotypes) thereof (for example,IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2), based on the identity of itsheavy chain constant domains, referred to as alpha, delta, epsilon,gamma, and mu, respectively. The different classes of immunoglobulinshave different and well-known subunit structures and three-dimensionalconfigurations. The term “antibody” does not refer to molecules that donot share homology with an immunoglobulin sequence. For example, theterm “antibody” as used herein does not include “repebodies”.

The term “antibody fragment” refers to a portion of an intact antibodyand refers to antigenic determining variable regions of an intactantibody. Examples of antibody fragments include Fab, Fab′, F(ab′)₂, Fd,and Fv fragments, linear antibodies, single chain antibodies, andmultispecific antibodies formed from antibody fragments.

The term “monoclonal antibody” refers to a homogeneous antibodypopulation involved in the highly specific recognition and binding of asingle antigenic determinant or epitope. This contrasts with polyclonalantibodies that typically include different antibodies directed againsta variety of different antigenic determinants. The term “monoclonalantibody” includes antibody fragments (such as Fab, Fab′, F(ab′)₂, Fd,Fv), single chain (scFv) mutants, fusion proteins including an antibodyportion, and any other modified immunoglobulin molecule including anantigen recognition site as well as both intact and full-lengthmonoclonal antibodies, but are not limited thereto. Additionally,“monoclonal antibody” refers to such antibodies made in any number ofmethods, including but not limited to hybridoma, phage selection,recombinant expression, and transgenic animals.

The term “humanized antibody” refers to forms of non-human (e.g.,murine) antibodies that are specific immunoglobulin chains, chimericimmunoglobulins, or fragments thereof that contain minimal non-human(e.g., murine) sequences. In general, humanized antibodies are humanimmunoglobulins in which residues from complementary determining region(CDR) are replaced by residues from CDR of a non-human species (e.g.,mouse, rat, rabbit, and hamster) having the desired specificity,affinity, and capability (see, e.g., Jones et al., Nature, 321:522-525(1986); Riechmann et al., Nature, 332:323-327 (1988); Verhoeyen et al.,Science, 239:1534-1536 (1988)). In some instances, Fv framework region(FR) residues of a human immunoglobulin are replaced with thecorresponding residues in an antibody from a non-human species having adesired specificity, affinity, and/or binding capability. The humanizedantibody may be further modified by the substitution of additionalresidues either in the Fv framework region and/or within the replacednon-human residues to refine and optimize antibody specificity,affinity, and/or binding capability. In general, a humanized antibodyincludes substantially all of at least one, and typically two or three,variable domains containing all or substantially all of the CDRs thatcorrespond to the non-human immunoglobulin whereas all or substantiallyall of the framework regions (FRs) have those of a human immunoglobulinconsensus sequence. The humanized antibody may also include at least aportion of an immunoglobulin constant region or domain (Fc), typicallythat of a human immunoglobulin. Examples of methods used to generatehumanized antibodies are described in U.S. Pat. No. 5,225,539, herebyincorporated by reference.

The term “human antibody” as used herein refers to an antibody encodedby a human nucleotide sequence or an antibody having an amino acidsequence corresponding to an antibody produced by a human using anytechnique known in the art. This definition of the human antibodyincludes intact full-length antibodies and/or fragments thereof.

The term “chimeric antibody” refers to an antibody wherein an amino acidsequence of an immunoglobulin molecule is derived from two or morespecies, one of which is preferably human. In general, variable regionsof both light and heavy chains correspond to variable regions ofantibodies derived from one species of mammals (e.g., mouse, rat,rabbit, etc.) with the desired specificity, affinity, and capability,while constant regions are homologous to the sequences in antibodiesderived from another species (usually human), e.g., to avoid elicitingan immune response in that species.

The terms “epitope” and “antigenic determinant” are used interchangeablyherein and refer to that portion of an antigen capable of beingrecognized and specifically bound by a particular antibody. When theantigen is or comprises a polypeptide or protein, epitopes may be formedfrom contiguous and/or non-contiguous amino acids, e.g., juxtaposed bysecondary, tertiary, and/or quaternary folding of a protein. Epitopesformed from contiguous amino acids are typically retained upon proteindenaturing, whereas epitopes formed by tertiary folding may be lost uponprotein denaturing. An epitope typically includes 3 or more, 5 or more,or 8 to 10 or more amino acids in a unique spatial conformation.

An antibody “specifically binds” to an epitope or antigenic molecule,which means that the antibody interacts or associates more frequently,more rapidly, with greater duration, with greater affinity, or with somecombination of the foregoing to an epitope or antigenic molecule thanalternative substances, including unrelated proteins. In specificembodiments, “specifically binds” means, for instance, that an antibodybinds to a protein with a K_(D) of about 0.1 mM or less, but moreusually, less than about 1 μM. In specific embodiments, “specificallybinds” means that an antibody binds to a protein at times with a K_(D)of about 0.1 μM or less, and at other times, with a K_(D) of about 0.01μM or less. Because of the sequence identity between homologous proteinsin different species, specific binding may include an antibodyrecognizing a particular protein in more than one species. It isunderstood that an antibody or binding residue that specifically bindsto a first target may or may not specifically bind to a second target.As described above, “specific binding” does not necessarily require(although it may include) exclusive binding, that is, binding to asingle target. Generally, but not necessarily, the term binding usedherein means specific binding.

The antibodies, including fragments/derivatives thereof and monoclonalantibodies, may be obtained using methods known in the art (see, e.g.,McCafferty et al., Nature 348:552-554 (1990); Clackson et al., Nature352:624-628; Marks et al., J. Mol. Biol. 222:581-597 (1991); Marks etal., Bio/Technology 10:779-783 (1992); Waterhouse et al., Nucleic AcidsRes. 21:2265-2266 (1993); Morimoto et al., J Biochemical & BiophysicalMethods 24:107-117 (1992); Brennan et al., Science 229:81(1985); Carteret al., Bio/Technology 10:163-167 (1992); Kohler et al., Nature 256:495(1975); Kilpatrick et al., Hybridoma 16(4):381-389 (1997); Wring et al.,J. Pharm. Biomed. Anal. 19(5):695-707 (1999); Bynum et al., Hybridoma18(5):407-411 (1999), Jakobovits et al., Proc. Natl. Acad. Sci. USA,90:2551 (1993); Jakobovits et al., Nature, 362:255-258 (1993);Bruggemann et al., Year Immuno. 7:33 (1993); Barbas et al., Proc. Nat.Acad. Sci. USA 91:3809-3813 (1994); Schier et al., Gene 169:147-155(1995); Yelton et al., J. Immunol. 155:1994-2004 (1995); Jackson et.al., J. Immunol. 154(7):3310-9 (1995); Hawkins et al., J. Mol. Biol.226:889-896 (1992), U.S. Pat. Nos. 4,816,567, 5,514,548, 5,545,806,5,569,825, 5,591,669, 5,545,807; PCT Patent Application Publication No.WO 97/17852, each of which is hereby incorporated by reference in itsentirety).

The antibody may be muromonab-CD3 abciximab, rituximab, daclizumab,palivizumab, infliximab, trastuzumab, etanercept, basiliximab,gemtuzumab, alemtuzumab, ibritumomab, adalimumab, alefacept, omalizumab,efalizumab, tositumomab, cetuximab, ABT-806, bevacizumab, natalizumab,ranibizumab, panitumumab, eculizumab, rilonacept, certolizumab,romiplostim, AMG-531, golimumab, ustekinumab, ABT-874, belatacept,belimumab, atacicept, an anti-CD20 antibody, canakinumab, tocilizumab,atlizumab, mepolizumab, pertuzumab, HuMax CD20, tremelimumab,ticilimumab, ipilimumab, IDEC-114, inotuzumab, HuMax EGFR, aflibercept,HuMax-CD4, teplizumab, otelixizumab, catumaxomab, the anti-EpCAMantibody IGN101, adecatumomab, oregovomab, dinutuximab, girentuximab,denosumab, bapineuzumab, motavizumab, efumgumab, raxibacumab, ananti-CD20 antibody, LY2469298, or veltuzumab.

When the antibody comprises at least one light chain and at least oneheavy chain, at least one light chain of the antibody, or at least oneheavy chain of the antibody, or both may comprise an amino acid regionhaving an amino acid motif capable of being recognized by an isoprenoidtransferase. As an antibody may comprise four polypeptide chains (e.g.,two heavy chains and two light chains), an antibody may comprise fouramino acid motifs, each of which can be used to conjugate an activeagent to the antibody via a linker. Thus, an antibody-drug conjugate maycomprise 4 linkers, each conjugated to at least one active agent.Accordingly, an antibody-drug conjugate may comprise at least one linkerand at least two active agents. An antibody-drug conjugate may compriseat least two linkers, and an antibody-drug conjugate may comprise atleast three active agents. An antibody-drug conjugate may comprise 1, 2,3, or 4 linkers. An antibody-drug conjugate may comprise 1, 2, 3, or 4peptides. An antibody-drug conjugate may comprise 2 to 100 activeagents, such as 2 to 50 active agents, 2 to 20 active agents, 2 to 16active agents, 4 to 16 active agents, or 4 to 8 active agents.

The active agent may be a drug, toxin, affinity ligand, detection probe,or combination of any of the foregoing.

The active agent may be selected from erlotinib; bortezomib;fulvestrant; sutent; letrozole; imatinib mesylate; PTK787/ZK 222584;oxaliplatin; 5-fluorouracil; leucovorin; rapamycin (Sirolimus);lapatinib; lonafarnib; sorafenib; gefitinib; AG1478; AG1571; alkylatingagents (for example, thiotepa or cyclophosphamide); alkyl sulfonate (forexample, busulfan, improsulfan, or piposulfan); aziridine (for example,benzodopa, carboquone, meturedopa, or uredopa); ethyleneimine,methylmelamine, altretamine, triethylenemelamine,triethylenephosphoramide, triethylenethiophosphoramide,trimethylolmelamine; acetogenins (for example, bullatacin orbullatacinone); camptothecin; topotecan; bryostatin; callystatin;CC-1065 (including its adozelesin, carzelesin, or bizelesin syntheticanalogs); cryptophycins (for example, cryptophycin 1 or cryptophycin 8);dolastatin; duocarmycin (including synthetic analogs, e.g., KW-2189 andCB1-TM1); eleutherobin; pancratistatin; sarcodictyin; spongistatin;nitrogen mustard (for example, chlorambucil, chlornaphazine,chlorophosphamide, estramustine, ifosfamide, mechlorethamine,mechlorethamine oxide hydrochloride, melphalan, novembichin,phenesterine, prednimustine, trofosfamide, or uracil mustard);nitrousurea (for example, carmustine, chlorozotocin, fotemustine,lomustine, nimustine, or ranimnustine); antibiotics (for example,enediyne antibiotics such as calicheamicin selected from calicheamicingamma 1I and calicheamicin omega 1I, or dynemicin including dynemicinA); bisphosphonate (for example, clodronate; esperamicin,neocarzinostatin chromophore, or related chromoprotein enediyneantibiotic chromophores, aclacinomysins, actinomycin, anthramycin,azaserine, bleomycins, cactinomycin, carabicin, carninomycin,carzinophilin, chromomycins, dactinomycin, daunorubicin, detorubucin,6-diazo-5-oxo-L-norleucine, doxorubicin (for example,morpholino-doxorubicin, cyanomorpholino-doxorubicin,2-pyrrolino-doxorubucin, liposomal doxorubicin, or deoxydoxorubicin),epirubicin, esorubicin, marcellomycin, mitomycins (for example,mitomycin C, mycophenolic acid, nogalamycin, olivomycins, peplomycin,potfiromycin, puromycin, quelamycin, rodorubicin, streptomigrin,streptozocin, tubercidin, ubenimex, zinostatin, or zorubicin);anti-metabolites (for example, 5-fluorouracil); folic acid analogs (forexample, denopterin, methotrexate, pteropterin, or trimetrexate); purineanalogs (for example, fludarabine, 6-mercaptopurine, thiamiprine, orthiguanine); pyrimidine analogs (for example, ancitabine, azacitidine,6-azauridine, carmofur, cytarabine, dideoxyuridine, doxifluridine,enocitabine, or floxuridine); androgens (for example, calusterone,dromostanolone propionate, epitiostanol, mepitiostane), ortestolactone); anti-adrenals (for example, aminoglutethimide, mitotane,or trilostane); folic acid replenisher (for example, folinic acid);aceglatone; aldophosphamide glycoside; aminolevulinic acid; eniluracil;amsacrine; bestrabucil; bisantrene; edatraxate; defofamine; demecolcine;diaziquone; elfornithine; elliptinium acetate; epothilone; etoglucid;gallium nitrate; hydroxyurea; lentinan; lonidainine; maytansinoids (forexample, maytansine or ansamitocins); trichothecenes (particularly T-2toxin, verracurin A, roridin A, or anguidine); mitoguazone;mitoxantrone; mopidanmol; nitraerine; pentostatin; phenamet;pirarubicin; losoxantrone; 2-ethylhydrazide; procarbazine;polysaccharide K complex; razoxane; rhizoxin; sizofiran; spirogermanium;tenuazonic acid; triaziquone; 2,2′,2″-trichlorotriethylamine;trichothecenes (particularly, T-2 toxin, verracurin A, roridin A, andanguidine); urethane; vindesine; dacarbazine; mannomustine;mitobronitol; mitolactol; pipobroman; gacytosine; arabinoside;cyclophosphamide; thiotepa; taxoids (for example, paclitaxel),ABRAXANErm cremophor-free, albumin-engineered nanoparticle formulationof paclitaxel, doxetaxel; chlorambucil; gemcitabine; 6-thioguanine;mercaptopurine; platinum analog (for example, cisplatin or carboplatin);vinblastine; platinum; etoposide, ifosfamide; mitoxantrone; vincristine;vinorelbine; novantrone; teniposide; edatrexate; daunomycin;aminopterin; xeloda; ibandronate; CPT-11; topoisomerase inhibitor (RFS2000); difluoromethylornithine; retinoid (for example, retinoic acid);capecitabine, and pharmaceutically acceptable salts, solvates, acids, orderivatives thereof, but is not necessarily limited thereto.

The active agent may be selected from (i) anti-hormonal agents that actto regulate or inhibit hormone action on tumors such as anti-estrogensand selective estrogen receptor modulators, including, for example,tamoxifen, raloxifene, droloxifene, 4-hydroxytamoxifen, trioxifene,keoxifene, LY117018, onapristone, and toremifene; (ii) aromataseinhibitors that inhibit aromatase enzyme, which regulates estrogenproduction in the adrenal glands, for example, 4(5)-imidazoles,aminoglutethimide, megestrol acetate, exemestane, letrozole, andanastrozole; (iii) anti-androgens such as flutamide, nilutamide,bicalutamide, leuprolide, and goserelin; as well as troxacitabine (a1,3-dioxolane nucleoside cytosine analog); (iv) aromatase inhibitors;(v) protein kinase inhibitors; (vi) lipid kinase inhibitors; (vii)antisense oligonucleotides, particularly those that inhibit expressionof genes in signaling pathways implicated in adherent cells, forexample, PKC-alpha, Raf, H-Ras; (viii) ribozyme, for example, VEGFinhibitor such as ribozyme and HER2 expression inhibitors; (ix) vaccinessuch as a gene therapy vaccine; ALLOVECTIN® vaccine, LEUVECTIN vaccine,VAXID vaccine; PROLEUKIN®rlL-2; LURTOTECAN® topoisomerase 1 inhibitor;ABARELIX® rmRH; (x) an anti-angiogenic agent such as Bevacizumab; and(xi) pharmaceutically acceptable salts, solvates, acids, or derivativesthereof.

In addition, cytokines may be used as the active agent. Cytokines aresmall cell-signaling protein molecules that are secreted by numerouscells and are a category of signaling molecules used extensively inintercellular communication. The cytokines include monokines,lymphokines, traditional polypeptide hormones, and the like. Examples ofthe cytokines include growth hormone (for example, human growth hormone,N-methionyl human growth hormone, or bovine growth hormone); parathyroidhormone; thyroxine; insulin; proinsulin; relaxin; prorelaxin;glycoprotein hormone (for example, follicle stimulating hormone (FSH),thyroid stimulating hormone (TSH), or luteinizing hormone (LH)); hepaticgrowth factor; fibroblast growth factor; prolactin; placental lactogen;tumor necrosis factor-α, tumor necrosis factor-β; mullerian-inhibitingsubstance; mouse gonadotropin-associated peptide; inhibin; activin;vascular endothelial growth factor; integrin, thrombopoietin (TPO);nerve growth factor (for example, NGF-β); platelet-growth factor;transforming growth factor (TGF) (for example, TGF-α or TGF-β);insulin-like growth factor-I, insulin-like growth factor-II;erythropoietin (EPO); osteoinductive factor; interferon (for example,interferon-α, interferon-β, or interferon-γ); colony stimulating factor(CSF) (for example, macrophage-CSF (M-CSF), granulocyte-macrophage-CSF(GM-CSF), or granulocyte-CSF (G-CSF)); interleukin (IL) (for example,IL-1, IL-1α, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10,IL-11, or IL-12); tumor necrosis factor (TNF) (for example, TNF-α orTNF-β); and polypeptide factor (for example, LIF or kit ligand), but arenot limited thereto. Further, the term “cytokine” also includescytokines from natural sources or recombinant cell cultures andbiologically active equivalents of the native sequence cytokines.

The term “toxin” refers substances that are poisonous to living cells ororganisms. Toxins may be small molecules, peptides or proteins capableof causing cell dysfunction or cell death after contact with orabsorption by body tissue, e.g., through an interaction with one or morebiological macromolecules such as enzymes or cell receptors. Toxinsinclude plant toxins and animal toxins. Examples of animal toxinsinclude diphtheria toxin, botulinum toxin, tetanus toxin, dysenterytoxin, cholera toxin, tetrodotoxin, brevetoxin, and ciguatoxin, but arenot limited thereto. Examples of plant toxins include ricin andAM-toxin, but are not limited thereto.

Examples of small molecule toxins include auristatin, tubulysin,geldanamycin (Kerr et al., 1997, Bioconjugate Chem. 8(6):781-784),maytansinoid (EP 1391213, ACR 2008, 41, 98-107), calicheamicin (U.S.Patent Publication No. 2009/0105461, Cancer Res. 1993, 53, 3336-3342),daunomycin, doxorubicin, methotrexate, vindesine, SG2285 (Cancer Res.2010, 70(17), 6849-6858), dolastatin, dolastatin analogs auristatin(U.S. Pat. No. 5,635,483), cryptophycin, camptothecin, rhizoxinderivative, CC-1065 analog or derivative, duocarmycin, enediyneantibiotic, esperamicin, epothilone, pyrrolobenzodiazepine (PBD)derivatives, α-amanitin, and toxoid, but are not limited thereto. Toxinsmay exhibit cytotoxicity and cell growth-inhibiting activity by tubulinbinding, DNA binding, topoisomerase suppression, and the like.

The term “ligand” refers to a molecule capable of forming a complex witha target biomolecule. An example of the ligand is a molecule bound to apredetermined position of a target protein to transmit a signal. Theligand may be a substrate, an inhibitor, a stimulating agent, aneurotransmitter, or a radioisotope.

“Detectable moiety” or a “label” refers to a composition detectable byspectroscopic, photochemical, biochemical, immunochemical, radioactive,or chemical means. For example, useful labels include ³²P, ³⁵S,fluorescent dyes, electron-dense reagents, enzymes (for example, enzymescommonly used in an ELISA), biotin-streptavidin, dioxigenin, haptens,and proteins for which antisera or monoclonal antibodies are available,or nucleic acid molecules with a sequence complementary to a target. Thedetectable moiety often generates a measurable signal, such as aradioactive, chromogenic, or fluorescent signal, that may be used toquantify the amount of bound detectable moiety in a sample. Quantitationof the signal may be achieved, for example, by scintillation counting,densitometry, flow cytometry, ELISA, or direct analysis by massspectrometry of intact or subsequently digested peptides (one or morepeptide may be assessed).

The term “probe” as used herein refers to a material that may (i)provide a detectable signal, (ii) interact a first probe or a secondprobe to modify a detectable signal provided by the first or secondprobe, such as fluorescence resonance energy transfer (FRET), (iii)stabilize an interaction with an antigen or a ligand or increase bindingaffinity; (iv) affect electrophoresis mobility or cell-intrudingactivity by a physical parameter such as charge, hydrophobicity, etc.,or (v) control ligand affinity, antigen-antibody binding, or ioniccomplex formation.

The active agent may be an immunomodulatory compound, an anticanceragent, an antiviral agent, an antibacterial agent, an antifungal agent,an antiparasitic agent, or a combination thereof.

An immunomodulatory compound may be selected from aminocaproic acid,azathioprine, bromocriptine, chlorambucil, chloroquine,cyclophosphamide, cyclosporine, cyclosporine A, danazol,dehydroepiandrosterone, dexamethasone, etanercept, hydrocortisone,hydroxychloroquine, infliximab, meloxicam, methotrexate, mycophenylatemofetil, prednisone, sirolimus, and tacrolimus. An anticancer agent maybe selected from 1-methyl-4-phenylpyridinium ion,5-ethynyl-1-beta-D-ribofuranosylimidazole-4-carboxamide (EICAR),5-fluorouracil, 9-aminocamptothecin, actinomycin D, asparaginase,bicalutamide, bis-chloroethylnitrosourea (BCNU), bleomycin, bleomycinA2, bleomycin B₂, busulfan, camptothecin, carboplatin, carmustine,CB1093, chlorambucil, cisplatin, crisnatol, cyclophosphamide,cytarabine, cytosine arabinoside, cytoxan, dacarbazine, dactinomycin,daunorubicin, decarbazine, deferoxamine, demethoxy-hypocrellin A,docetaxel, doxifluridine, doxorubicin, EB1089, epirubicin, etoposide,floxuridine, fludarabine, flutamide, gemcitabine, goserelin,hydroxyurea, idarubicin, ifosfamide, interferon-α, interferon-γ,irinotecan, KH1060, leuprolide acetate, lomustine, lovastatin,megestrol, melphalan, mercaptopurine, methotrexate, mitomycin, mitomycinC, mitoxantrone, mycophenolic acid, nitrogen mustard, nitrosourea,paclitaxel, peplomycin, photosensitizer Pe4, phthalocyanine,pirarubicin, plicamycin, procarbazine, raloxifene, raltitrexed,revlimid, ribavirin, staurosporine, tamoxifen, teniposide, thalomid,thapsigargin, thioguanine, tiazofurin, topotecan, treosulfan,trimetrexate, tumor necrosis factor, velcade, verapamil, verteporfin,vinblastine, vincristine, vinorelbine, and zorubicin. An antiviral agentmay be selected from pencicyclovir, valacyclovir, gancicyclovir,foscarnet, ribavirin, idoxuridine, vidarabine, trifluridine, acyclovir,famcicyclovir, amantadine, rimantadine, cidofovir, antisenseoligonucleotide, immunoglobulin, and interferon. An antibacterial agentmay be selected from chloramphenicol, vancomycin, metronidazole,trimethoprin, sulfamethazole, quinupristin, dalfopristin, rifampin,spectinomycin, and nitrofurantoin. The antifungal agent may be selectedfrom amphotericin B, candicidin, filipin, hamycin, natamycin, nystatin,rimocidin, bifonazole, butoconazole, clotrimazole, econazole,fenticonazole, isoconazole, ketoconazole, luliconazole, miconazole,omoconazole, oxiconazole, sertaconazole, sulconazole, tioconazole,albaconazole, fluconazole, isavuconazole, itraconazole, posaconazole,ravuconazole, terconazole, voriconazole, abafungin, amorolfin,butenafine, naftifine, terbinafine, anidulafungin, caspofungin,micafungin, benzoic acid, ciclopirox, flucytosine, griseofulvin,haloprogin, tolnaftate, undecylenic acid, crystal violet, balsam ofperu, ciclopirox olamine, piroctone olamine, zinc pyrithione, andselenium sulfide. An antiparasitic agent may be selected frommebendazole, pyrantel pamoate, thiabendazole, diethylcarbamazine,ivermectin, niclosamide, praziquantel, albendazole, rifampin,amphotericin B, melarsoprol, eflornithine, metronidazole, tinidazole,and miltefosine.

The antibody may comprise an amino acid motif selected fromAb-HC-(G)zCVIM, Ab-HC-(G)zCVLL, Ab-LC-(G)zCVIM, and Ab-LC-(G)zCVLL,wherein Ab represents an antibody, —HC— represents a heavy chain, -LC-represents a light chain, G represents a glycine, C represents cysteine,V represents valine, I represents isoleucine, M represents methionine, Lrepresents leucine, and z is an integer from 0 to 20.

B, B₁, and/or B₂ (e.g., of Formulas (I) and (V)-(IX)) may eachindependently be selected from any one of the following structures:

wherein y is an integer from 1 to 10.

The antibody-drug conjugate may be used to transfer the active agent toa target cell of a subject to treat the subject using a method ofpreparing a composition known to those skilled in the art. In someaspects, the invention relates to a composition (e.g., a pharmaceuticalcomposition) comprising an antibody-drug conjugate as described herein.

Compositions may be prepared in an injectable form, either as a liquidsolution or as a suspension. Solid forms suitable for injection may alsobe prepared, e.g., as emulsions, or with the antibody-drug conjugateencapsulated in liposomes. Antibody-drug conjugates may be combined witha pharmaceutically acceptable carrier, which includes any carrier thatdoes not induce the production of antibodies harmful to the subjectreceiving the carrier. Suitable carriers typically comprise largemacromolecules that are slowly metabolized, for example, proteins,polysaccharides, polylactic acids, polyglycolic acids, polymeric aminoacids, amino acid copolymers, lipid aggregates, and the like.

The compositions may also contain diluents, for example, water, saline,glycerol, and ethanol. Auxiliary substances, for example, wetting oremulsifying agents, pH buffering substances, and the like may also bepresent therein. The compositions may be parenterally administered byinjection, wherein such injection may be either subcutaneous orintramuscular injection. In some embodiments, a composition may beadministered into a tumor. The composition may be inserted (e.g.,injected) into a tumor. Additional formulations are suitable for otherforms of administration, such as suppository or oral administration.Oral compositions may be administered as a solution, suspension, tablet,pill, capsule, or sustained release formulation.

The compositions may be administered in a manner compatible with a doseand a formulation. The composition preferably comprises atherapeutically effective amount of the antibody-drug conjugate. Theterm “therapeutically effective amount” means a single dose or acomposition administered in a multiple dose schedule that is effectivefor the treatment or prevention of a disease or disorder. A dose mayvary, depending on the subject to be treated, the subject's health andphysical conditions, a degree of protection to be desired, and otherrelevant factors. The exact amount of an active ingredient (e.g., theantibody-drug conjugate) may depend on the judgment of a doctor. Forexample, a therapeutically effective amount of the antibody-drugconjugate or composition containing the same may be administered to apatient suffering from a cancer or tumor to treat the cancer or tumor.

The antibody-drug conjugate according to the present invention or thecomposition containing the same may be administered in the form of apharmaceutically acceptable salt or solvate thereof. In someembodiments, the antibody-drug conjugate according to the presentinvention or the composition containing the same may be administeredwith a pharmaceutically acceptable carrier, a pharmaceuticallyacceptable excipient, and/or a pharmaceutically acceptable additive. Theeffective amount and the type of the pharmaceutically acceptable salt orsolvate, excipient and additive may be measured using standard methods(see, e.g., Remington's Pharmaceutical Sciences, Mack Publishing Co.,Easton, Pa., 18th Edition, 1990).

The term “therapeutically effective amount” with regard to cancer ortumor means an amount that may decrease the number of cancer cells;decrease a size of cancer cells; inhibit cancer cells from intrudinginto peripheral systems or decrease the intrusion; inhibit cancer cellsfrom spreading to other systems or decrease the spreading; inhibitcancer cells from growing; and/or ameliorate at least one symptomrelated to the cancer. In the treatment of cancer, the effectiveness ofa drug may be assessed by time to tumor progression (TTP) and/orresponse rate (RR).

The term “pharmaceutically acceptable salts” used herein includesorganic salts and inorganic salts. Examples thereof includehydrochloride, hydrobromide, hydroiodide, sulfate, citrate, acetate,oxalate, chloride, bromide, iodide, nitrate, bisulfate, phosphate,acidic phosphate, isonicotinate, lactate, salicylate, acidic citrate,tartrate, oleate, tannate, pantonate, bitartrate, ascorbate, succinate,maleate, gentisinate, fumarate, gluconate, glucoronate, saccharate,formate, benzoate, glutamate, methane sulfonate, ethane sulfonate,benzene sulfonate, p-toluene sulfonate, and pamoate (that is,1,1′-methylenebis-(2-hydroxy-3-naphthoate)). The pharmaceuticallyacceptable salt may include another molecule (for example, acetate ions,succinate ions, and/or other counter ions).

Exemplary solvates that may be used for pharmaceutically acceptablesolvates of the antibody-drug conjugates described herein include water,isopropanol, ethanol, methanol, dimethyl sulfoxide, ethyl acetate,acetic acid, and ethanol amine. The term “acyl” is art-recognized andrefers to a group represented by the general formula hydrocarbylC(O)—,preferably alkylC(O)—.

The term “acylamino” is art-recognized and refers to an amino groupsubstituted with an acyl group and may be represented, for example, bythe formula hydrocarbylC(O)NH—.

The term “acyloxy” is art-recognized and refers to a group representedby the general formula hydrocarbylC(O)O—, preferably alkylC(O)O—.

The term “alkoxy” refers to an alkyl group, preferably a lower alkylgroup, having an oxygen attached thereto. Representative alkoxy groupsinclude methoxy, ethoxy, propoxy, tert-butoxy and the like.

The term “alkoxyalkyl” refers to an alkyl group substituted with analkoxy group and may be represented by the general formulaalkyl-O-alkyl.

The term “alkenyl”, as used herein, refers to an aliphatic groupcontaining at least one double bond and is intended to include both“unsubstituted alkenyls” and “substituted alkenyls”, the latter of whichrefers to alkenyl moieties having substituents replacing a hydrogen onone or more carbons of the alkenyl group. Such substituents may occur onone or more carbons that are included or not included in one or moredouble bonds. Moreover, such substituents include all those contemplatedfor alkyl groups, as discussed below, except where stability isprohibitive. For example, substitution of alkenyl groups by one or morealkyl, carbocyclyl, aryl, heterocyclyl, or heteroaryl groups iscontemplated.

An “alkyl” group or “alkane” is a straight chained or branchednon-aromatic hydrocarbon which is completely saturated. Typically, astraight chained or branched alkyl group has from 1 to about 20 carbonatoms, preferably from 1 to about 10 unless otherwise defined. Examplesof straight chained and branched alkyl groups include methyl, ethyl,n-propyl, iso-propyl, n-butyl, sec-butyl, tert-butyl, pentyl, hexyl,pentyl and octyl. A C₁-C₆ straight chained or branched alkyl group isalso referred to as a “lower alkyl” group.

Moreover, the term “alkyl” (or “lower alkyl”) as used throughout thespecification, examples, and claims is intended to include both“unsubstituted alkyls” and “substituted alkyls”, the latter of whichrefers to alkyl moieties having substituents replacing a hydrogen on oneor more carbons of the hydrocarbon backbone. Such substituents, if nototherwise specified, can include, for example, a halogen, a hydroxyl, acarbonyl (such as a carboxyl, an alkoxycarbonyl, a formyl, or an acyl),a thiocarbonyl (such as a thioester, a thioacetate, or a thioformate),an alkoxyl, a phosphoryl, a phosphate, a phosphonate, a phosphinate, anamino, an amido, an amidine, an imine, a cyano, a nitro, an azido, asulfhydryl, an alkylthio, a sulfate, a sulfonate, a sulfamoyl, asulfonamido, a sulfonyl, a heterocyclyl, an aralkyl, or an aromatic orheteroaromatic moiety. It will be understood by those skilled in the artthat the moieties substituted on the hydrocarbon chain can themselves besubstituted, if appropriate. For instance, the substituents of asubstituted alkyl may include substituted and unsubstituted forms ofamino, azido, imino, amido, phosphoryl (including phosphonate andphosphinate), sulfonyl (including sulfate, sulfonamido, sulfamoyl andsulfonate), and silyl groups, as well as ethers, alkylthios, carbonyls(including ketones, aldehydes, carboxylates, and esters), —CF₃, —CN andthe like. Exemplary substituted alkyls are described below. Cycloalkylscan be further substituted with alkyls, alkenyls, alkoxys, alkylthios,aminoalkyls, carbonyl-substituted alkyls, —CF₃, —CN, and the like.

The term “C_(x-y)” when used in conjunction with a chemical moiety, suchas, acyl, acyloxy, alkyl, alkenyl, alkynyl, or alkoxy is meant toinclude groups that contain from x to y carbons in the chain. Forexample, the term “C_(x)-yalkyl” refers to substituted or unsubstitutedsaturated hydrocarbon groups, including straight-chain alkyl andbranched-chain alkyl groups that contain from x to y carbons in thechain, including haloalkyl groups such as trifluoromethyl and2,2,2-trifluoroethyl, etc. Co alkyl indicates a hydrogen where the groupis in a terminal position, a bond if internal. The terms“C_(2-y)alkenyl” and “C_(2-y)alkynyl” refer to substituted orunsubstituted unsaturated aliphatic groups analogous in length andpossible substitution to the alkyls described above, but that contain atleast one double or triple bond respectively.

The term “alkylamino”, as used herein, refers to an amino groupsubstituted with at least one alkyl group.

The term “alkylthio”, as used herein, refers to a thiol groupsubstituted with an alkyl group and may be represented by the generalformula alkylS-.

The term “alkynyl”, as used herein, refers to an aliphatic groupcontaining at least one triple bond and is intended to include both“unsubstituted alkynyls” and “substituted alkynyls”, the latter of whichrefers to alkynyl moieties having substituents replacing a hydrogen onone or more carbons of the alkynyl group. Such substituents may occur onone or more carbons that are included or not included in one or moretriple bonds. Moreover, such substituents include all those contemplatedfor alkyl groups, as discussed above, except where stability isprohibitive. For example, substitution of alkynyl groups by one or morealkyl, carbocyclyl, aryl, heterocyclyl, or heteroaryl groups iscontemplated.

The term “amide”, as used herein, refers to a group

wherein each R¹⁰ independently represent a hydrogen or hydrocarbylgroup, or two R¹⁰ are taken together with the N atom to which they areattached complete a heterocycle having from 4 to 8 atoms in the ringstructure.

The terms “amine” and “amino” are art-recognized and refer to bothunsubstituted and substituted amines and salts thereof, e.g., a moietythat can be represented by

wherein each R¹⁰ independently represents a hydrogen or a hydrocarbylgroup, or two R¹⁰ are taken together with the N atom to which they areattached complete a heterocycle having from 4 to 8 atoms in the ringstructure.

The term “aminoalkyl”, as used herein, refers to an alkyl groupsubstituted with an amino group.

The term “carboxy”, as used herein, refers to a group represented by theformula —CO₂H.

The terms “hetaralkyl” and “heteroaralkyl”, as used herein, refers to analkyl group substituted with a hetaryl group.

The term “heteroalkyl”, as used herein, refers to a saturated orunsaturated chain of carbon atoms and at least one heteroatom, whereinno two heteroatoms are adjacent.

The terms “heteroaryl” and “hetaryl” include substituted orunsubstituted aromatic single ring structures, preferably 5- to7-membered rings, more preferably 5- to 6-membered rings, whose ringstructures include at least one heteroatom, preferably one to fourheteroatoms, more preferably one or two heteroatoms. The terms“heteroaryl” and “hetaryl” also include polycyclic ring systems havingtwo or more cyclic rings in which two or more carbons are common to twoadjoining rings wherein at least one of the rings is heteroaromatic,e.g., the other cyclic rings can be cycloalkyls, cycloalkenyls,cycloalkynyls, aryls, heteroaryls, and/or heterocyclyls. Heteroarylgroups include, for example, pyrrole, furan, thiophene, imidazole,oxazole, thiazole, pyrazole, pyridine, pyrazine, pyridazine, andpyrimidine, and the like.

The term “heteroatom” as used herein means an atom of any element otherthan carbon or hydrogen. Preferred heteroatoms are nitrogen, oxygen, andsulfur. The terms “heterocyclyl”, “heterocycle”, and “heterocyclic”refer to substituted or unsubstituted non-aromatic ring structures,preferably 3- to 10-membered rings, more preferably 3- to 7-memberedrings, whose ring structures include at least one heteroatom, preferablyone to four heteroatoms, more preferably one or two heteroatoms. Theterms “heterocyclyl” and “heterocyclic” also include polycyclic ringsystems having two or more cyclic rings in which two or more carbons arecommon to two adjoining rings wherein at least one of the rings isheterocyclic, e.g., the other cyclic rings can be cycloalkyls,cycloalkenyls, cycloalkynyls, aryls, heteroaryls, and/or heterocyclyls.Heterocyclyl groups include, for example, piperidine, piperazine,pyrrolidine, morpholine, lactones, lactams, and the like. Heterocyclylgroups can also be substituted by oxo groups. For example,“heterocyclyl” encompasses both pyrrolidine and pyrrolidinone.

The term “hydroxyalkyl”, as used herein, refers to an alkyl groupsubstituted with a hydroxy group.

The term “substituted” refers to moieties having substituents replacinga hydrogen on one or more carbons of the backbone. It will be understoodthat “substitution” or “substituted with” includes the implicit provisothat such substitution is in accordance with permitted valence of thesubstituted atom and the substituent, and that the substitution resultsin a stable compound, e.g., which does not spontaneously undergotransformation such as by rearrangement, cyclization, elimination, etc.As used herein, the term “substituted” is contemplated to include allpermissible substituents of organic compounds.

In a broad aspect, the permissible substituents include acyclic andcyclic, branched and unbranched, carbocyclic and heterocyclic, aromaticand non-aromatic substituents of organic compounds. The permissiblesubstituents can be one or more and the same or different forappropriate organic compounds. For purposes of this invention, theheteroatoms such as nitrogen may have hydrogen substituents and/or anypermissible substituents of organic compounds described herein whichsatisfy the valences of the heteroatoms. Substituents can include anysubstituents described herein, for example, a halogen, a hydroxyl, acarbonyl (such as a carboxyl, an alkoxycarbonyl, a formyl, or an acyl),a thiocarbonyl (such as a thioester, a thioacetate, or a thioformate),an alkoxyl, a phosphoryl, a phosphate, a phosphonate, a phosphinate, anamino, an amido, an amidine, an imine, a cyano, a nitro, an azido, asulthydryl, an alkylthio, a sulfate, a sulfonate, a sulfamoyl, asulfonamido, a sulfonyl, a heterocyclyl, an aralkyl, or an aromatic orheteroaromatic moiety. It will be understood by those skilled in the artthat substituents can themselves be substituted, if appropriate. Unlessspecifically stated as “unsubstituted,” references to chemical moietiesherein are understood to include substituted variants. For example,reference to an “aryl” group or moiety implicitly includes bothsubstituted and unsubstituted variants.

The term “thioalkyl”, as used herein, refers to an alkyl groupsubstituted with a thiol group.

The term “thioester”, as used herein, refers to a group —C(O)SR¹⁰ or—SC(O)R¹⁰ wherein R¹⁰ represents a hydrocarbyl.

The term “thioether”, as used herein, is equivalent to an ether, whereinthe oxygen is replaced with a sulfur.

“Protecting group” refers to a group of atoms that, when attached to areactive functional group in a molecule, mask, reduce or prevent thereactivity of the functional group. Typically, a protecting group may beselectively removed as desired during the course of a synthesis.Examples of protecting groups can be found in Greene and Wuts,Protective Groups in Organic Chemistry, 3rd Ed., 1999, John Wiley &Sons, NY and Harrison et al., Compendium of Synthetic Organic Methods,Vols. 1-8, 1971-1996, John Wiley & Sons, NY. Representative nitrogenprotecting groups include, but are not limited to, formyl, acetyl,trifluoroacetyl, benzyl, benzyloxycarbonyl (“CBZ”), tert-butoxycarbonyl(“Boc”), trimethylsilyl (“TMS”), 2-trimethylsilyl-ethanesulfonyl(“TES”), trityl and substituted trityl groups, allyloxycarbonyl,9-fluorenylmethyloxycarbonyl (“FMOC”), nitro-veratryloxycarbonyl(“NVOC”) and the like. Representative hydroxylprotecting groups include,but are not limited to, those where the hydroxyl group is eitheracylated (esterified) or alkylated such as benzyl and trityl ethers, aswell as alkyl ethers, tetrahydropyranyl ethers, trialkylsilyl ethers(e.g., TMS or TIPS groups), glycol ethers, such as ethylene glycol andpropylene glycol derivatives and allyl ethers.

“Covalently coupled” includes both direct bonding and indirect bonding(e.g., through an intervening series of atoms) of two chemical species.For example, an amino acid may be covalently coupled to polyethyleneglycol directly, e.g., by forming an ester between the carboxyl of theamino acid and a hydroxyl of the polyethylene glycol, or indirectly,e.g., by reacting the polyethylene glycol with epichlorohydrin to forman epoxypropyl ether and reacting the resulting epoxide with the aminogroup of the amino acid, thereby covalently linking the amino acid andthe polyethylene glycol through a 2-hydroxypropyl linker. Variousmoieties and reactions for coupling diverse moieties directly orindirectly are well known in the art. In certain preferred embodiments,unless otherwise indicated by the context, an indirect bonding involvesonly 1-10 intervening atoms (e.g., a methylene, a dibutyl ether, atripeptide, etc.), most preferably 1-6 intervening atoms.

As used herein, a therapeutic that “prevents” a disorder or conditionrefers to a compound that, in a statistical sample, reduces theoccurrence of the disorder or condition in the treated sample relativeto an untreated control sample, or delays the onset or reduces theseverity of one or more symptoms of the disorder or condition relativeto the untreated control sample.

The term “treating” includes prophylactic and/or therapeutic treatments.The term “prophylactic or therapeutic” treatment is art-recognized andincludes administration to the host of one or more of the subjectcompositions. If it is administered prior to clinical manifestation ofthe unwanted condition (e.g., disease or other unwanted state of thehost animal) then the treatment is prophylactic (i.e., it protects thehost against developing the unwanted condition), whereas if it isadministered after manifestation of the unwanted condition, thetreatment is therapeutic, (i.e., it is intended to diminish, ameliorate,or stabilize the existing unwanted condition or side effects thereof).

The term “prodrug” is intended to encompass compounds which, underphysiologic conditions, are converted into the therapeutically activeagents. A common method for making a prodrug is to include one or moreselected moieties which are hydrolyzed under physiologic conditions toreveal the desired molecule. In other embodiments, the prodrug isconverted by an enzymatic activity of the host animal. For example,esters or carbonates (e.g., esters or carbonates of alcohols orcarboxylic acids) are preferred prodrugs.

In some embodiments, the invention relates to a method of treating adisease, such as cancer, in a subject, comprising administering apharmaceutical composition comprising an antibody-drug conjugate asdescribed herein to the subject. A pharmaceutical composition mayfurther comprise a therapeutically effective amount of chemotherapeuticagent. In preferred embodiments, the subject is a mammal. For example,the subject may be selected from rodents, lagomorphs, felines, canines,porcines, ovines, bovines, equines, and primates. In certain preferredembodiments, the subject is a human.

Hereinafter, configurations of the present invention will be describedin detail through Examples, but the following Examples are only toassist in understanding of the present invention. The scope of thepresent invention is not limited thereto.

EXEMPLIFICATION

The table below lists the abbreviations used throughout the followingExamples:

Abbreviation Reference Ac acetyl AcOH acetic acid aq. aqueous Bn benzylbrine saturated aqueous sodium chloride solution Boc t-butoxycarbonylCbz benzyloxycarbonyl DBU 1,8-diazabicyclo[5.4.0]undec-7-ene DCMdichloromethane DIC N,N′-diisopropylcarbodiimide DIPEAN,N-diisopropylethylamine DMAP 4-(dimethylamino)pyridine DMFN,N-dimethylformamide DMSO dimethyl sulfoxide EDCN-(3-dimethylaminopropyl)-N′-ethylcarbodiimide Et ethyl Et2O diethylether EtOAc ethyl acetate EtOH ethanol HBTUO-(benzotriazol-1-yl)-N,N,N′,N′-tetramethyluronium hexafluorophosphateHex n-hexane HOBt 1-hydroxybenzotriazole HPLC high performance liquidchromatography Me Methyl MeCN acetonitrile MeOH methanol MMAE monomethylauristatin E MMAF monomethyl auristatin F MMAF- monomethyl auristatin Fmethyl ester OMe i-PrOH isopropanol PyBOP(benzotriazol-1-yloxy)tripyrrolidinophosphonium hexafluorophosphate TBAFtetrabutylammonium fluoride TBS t-butyldimethylsilyl THF tetrahydrofuranTFA trifluoroacetic acid Ts p-toluenesulfonyl wt weight

Example 1. Preparation of Compound 1i

Preparation of Compound 1b

To a suspension of 5-formylsalicylic acid 1a (10.0 g, 60.1 mmol) in THF(30 mL) was added DIPEA (29.8 mL, 180 mmol) and benzyl bromide (7.15 mL,60.1 mmol) at room temperature. Then the reaction mixture was heatedunder reflux. After 18 hours under reflux, the reaction mixture dilutedwith 2 N aq. HCl (100 mL). The resulting mixture was extracted withEtOAc (2×100 mL). The combined organic layers were dried over anhydrousMgSO₄, filtered and concentrated. The residue was purified by columnchromatography to produce the compound 1b (12.9 g, 83%). ¹H-NMR (400MHz, CDCl₃) δ 11.38 (s, 1H), 9.86 (s, 1H), 8.40 (s, 1H), 8.01 (d, J=8.8Hz, 1H), 7.44 (m, 5H), 7.12 (d, J=8.0 Hz, 1H), 5.42 (s, 2H).

Preparation of Compound 1c

To a solution of compound 1b (5.0 g, 19.5 mmol) and compound M (8.5 g,21.4 mmol, see Example 66) in MeCN (100 mL) were added 4 Å molecularsieve (10 g) and Ag₂O (18.0 g, 78.0 mmol). After stirring at roomtemperature for 12 hours under N₂, the reaction mixture wasconcentrated. Then the concentrated reaction mixture was diluted withH₂O (100 mL) and extracted with EtOAc (2×200 mL). The combined organiclayers were dried over anhydrous MgSO₄, filtered and concentrated. Theresidue was purified by column chromatography to produce the compound 1c(8.63 g, 77%). ¹H-NMR (400 MHz, CDCl₃) δ 9.94 (s, 1H), 8.28 (s, 1H),8.02 (d, J=8.8 Hz, 1H), 7.46-7.28 (m, 6H), 5.41-5.32 (m, 6H), 4.27 (d,J=9.2 Hz, 1H), 3.71 (s, 3H), 2.05 (m, 9H).

Preparation of Compound 1d

To a solution of compound 1c (3.10 g, 5.41 mmol) in i-PrOH/CHCl₃ (9mL/45 mL) was added silica gel (3 g) and NaBH₄ (0.41 g, 10.82 mmol) at0° C. After stirring at 0° C. for 2 hours under N₂, the reaction mixturewas quenched with H₂O (100 mL) and extracted with EtOAc (200 mL). Theorganic layer was dried over anhydrous MgSO₄, filtered and concentrated.The crude product was purified by column chromatography to produce thecompound 1d (2.73 g, 87%) as white solid. ¹H-NMR (400 MHz, CDCl₃) δ 7.74(s, 1H), 7.48-7.34 (m, 6H), 7.16 (d, J=8.8 Hz, 1H), 5.35-5.26 (m, 5H),5.15 (m, 1H), 4.17 (m, 1H), 3.73 (s, 3H), 2.04 (s, 9H), 1.73 (t, 1H).

Preparation of Compound 1e

To a solution of compound 1d (2.40 g, 4.17 mmol) in EtOH (150 mL) Pd/C(10 wt. %, 240 mg) was added. The reaction mixture was stirred at roomtemperature for 10 minutes under hydrogen. Then the reaction mixture wasfiltered through a celite pad and washed with EtOH (100 mL). Thefiltrate was concentrated to provide the crude product 1e as white solid(2.10 g), which was used without further purification. ¹H-NMR (400 MHz,CDCl₃) δ 8.06 (s, 1H) 7.61 (dd, J=8.8 Hz, 1H), 7.23 (d, J=8.0 Hz 1H),5.43-5.29 (m, 5H), 4.17 (s, 2H), 4.32 (d, J=8.4 Hz, 1H) 3.69 (s, 3H),2.11-2.08 (t, 9H), 1.24 (t, 1H).

Preparation of Compound 1f

To a solution of the crude compound 1e (2.10 g, 4.33 mmol) in DMF (50mL) were added K₂CO₃ (1.79 g, 13.01 mmol) and allyl bromide (0.41 mL,4.76 mmol) at room temperature. After stirring at room temperature for 3hours, the reaction mixture was diluted with 2 N aq. HCl (100 mL). Theresulting mixture was extracted with EtOAc (200 mL). The organic layerwas dried over anhydrous MgSO₄, filtered and concentrated. The residuewas purified by column chromatography to produce the compound 1f (1.55g, 70% for 2 steps). ¹H-NMR (400 MHz, CDCl₃) δ 7.74 (s, 1H), 7.45 (dd,J=8.0 Hz, 2.0 Hz, 1H), 7.16 (d, J=8.8 Hz, 1H), 6.02 (m, 1H), 5.40-5.26(m, 5H), 5.16 (m, 1H), 4.76 (m, 2H), 4.66 (s, 2H), 4.19 (m, 1H), 3.73(s, 3H), 2.07-2.05 (m, 9H), 1.68 (t, 1H).

Preparation of Compound 1g

To a solution of compound 1f (2.50 g, 4.77 mmol) in DMF (20 mL) wereadded bis(4-nitrophenyl)carbonate (1.30 g, 4.29 mmol) and DIPEA (0.80 mL4.77 mmol) at 0° C. under N₂. The reaction mixture was stirred at 0° C.for 30 min and allowed to warm to room temperature for 1 hour. Thereaction mixture was diluted with H₂O (100 mL) and extracted with EtOAc(200 mL). The organic layer was washed with brine (100 mL) and driedover anhydrous MgSO₄. After filtration and concentration under reducedpressure, the resulting crude product was purified by columnchromatography to produce the compound 1g (2.80 g, 85%). ¹H-NMR (400MHz, CDCl₃) δ 8.28 (d, J=15.2 Hz, 2H), 7.85 (d, J=2.4 Hz, 1H), 7.55 (dd,J=3.2 Hz, 2.4 Hz, 1H), 7.38 (d, J=15.2 Hz, 2H), 7.20 (d, J=8.8 Hz, 1H)6.03 (m, 1H), 5.42-5.19 (m, 8H), 4.78 (d, J=5.2 Hz, 2H), 4.12 (d, J=7.2Hz, 1H), 3.74 (s, 3H).

Preparation of Compound 1h

Compound 1g (528 mg, 0.77 mmol), MMAE (500 mg, 0.7 mmol) and anhydrousHOBt (19 mg, 0.14 mmol) were dissolved in DMF (3 mL) at 0° C. Thenpyridine (0.7 mL) and DIPEA (0.24 mL, 1.39 mmol) were added. Afterstirring at room temperature for 24 hours under N₂, the reaction mixturewas diluted with H₂O/saturated aqueous NH₄Cl solution (100 mL/50 mL) andextracted with EtOAc (2×100 mL). The combined organic layers were driedover anhydrous MgSO₄, filtered, and concentrated. The residue waspurified by column chromatography to produce the compound 1h (600 mg,67%). EI-MS m/z: [M+H]⁺ 1269.5, [M+Na]⁺ 1291.5.

Preparation of Compound 1i

To a solution of compound 1h (600 mg, 0.47 mmol) and triphenylphosphine(31 mg, 0.12 mmol) in DCM (10 mL) were added pyrrolidine (0.047 mL, 0.57mmol) and Pd(PPh₃)₄ (27 mg, 0.02 mmol) at room temperature. Afterstirring for 2 hours, the reaction mixture was diluted with H₂O/1 N aq.HCl (50 mL/50 mL) and extracted with EtOAc (3×50 mL). The combinedorganic layers were dried over anhydrous MgSO₄, filtered, andconcentrated. The residue was purified by column chromatography(Hex/EtOAc 1/1 to EtOAc) to produce the compound 1i (480 mg, 82%) as awhite solid. EI-MS m/z: [M+H]⁺ 1228.4, [M+Na]⁺ 1250.4.

Example 2. Preparation of Compound 1j

Compound 1j was prepared from MMAF-OMe by a similar method of preparingcompound 1i in Example 1.

Example 3. Preparation of Compound 2g

Preparation of Compound 2a

2-(2-(2-Chloroethoxy)ethoxy)ethanol (10 g, 59.3 mmol) was dissolved inDMF (90 mL) at room temperature under nitrogen, and then NaN₃ (5.78 g,88.9 mmol) was added thereto. After stirring at 100° C. for 13 hours,chloroform (200 mL) and distilled water (300 mL) were added thereto toextract an organic layer, and the extracted organic layer was dried overanhydrous Na₂SO₄ and concentrated under reduced pressure. The residuewas subjected to column chromatography, which produced the compound 2a(10.3 g, 99%). ¹H-NMR (600 MHz, CDCl₃) δ 3.75-3.73 (m, 2H), 3.70-3.68(m, 6H), 3.63-3.61 (m, 2H), 3.40 (t, J=5.4 Hz, 2H), 2.20 (t, J=6.0 Hz,1H).

Preparation of Compound 2b

CBr₄ (21.4 g, 64.6 mmol) was dissolved in DCM (100 mL) at 0° C. undernitrogen, and then triphenylphosphine (16.9 g, 64.6 mmol) in DCM (100mL) and compound 2a (10.3 g, 58.7 mmol) were added thereto, and themixture was stirred at room temperature for 13 hours. After the reactionwas completed, DCM (300 mL) and distilled water (300 mL) were addedthereto to extract an organic layer, and the extracted organic layer wasdried over anhydrous Na₂SO₄ and concentrated under reduced pressure. Theresidue was subjected to column chromatography, which produced thecompound 2b (12 g, 85%). ¹H-NMR (400 MHz, CDCl₃) δ 3.83 (t, J=6.4 Hz,2H), 3.72-3.67 (m, 6H), 3.48 (t, J=6.0 Hz, 2H), 3.40 (t, J=4.8 Hz, 2H)

Preparation of Compound 2c

Compound 2b (1 g, 4.20 mmol) was dissolved in MeCN at room temperatureunder nitrogen, and then N-Boc-hydroxylamine (643 mg, 4.82 mmol) and DBU(0.66 mL, 4.41 mmol) were added thereto. After stirring at 60° C. for 13hours, DCM (300 mL) and distilled water (300 mL) were added thereto toextract an organic layer, and the extracted organic layer was dried overanhydrous Na₂SO₄ and concentrated under reduced pressure. The residuewas subjected to column chromatography, which produced the compound 2c(748 mg, 70%). ¹H-NMR (400 MHz, CDCl₃) δ 7.55 (s, 1H), 4.05-4.03 (m,2H), 3.76-3.74 (m, 2H), 3.74-3.69 (m, 6H), 3.42 (t, J=4.8 Hz, 2H), 1.49(s, 9H).

Preparation of Compound 2d

Compound 2c (200 mg, 0.688 mmol) was dissolved in MeOH (5 mL), and thenPd/C (10% wt., 70 mg) was added thereto and stirred under hydrogen for 3hours. After the reaction was completed, the reaction mixture wascelite-filtered and concentrated under reduced pressure, which producedthe compound 2d (180 mg, 98%). ¹H-NMR (400 MHz, CDCl₃) δ 4.04-4.01 (m,2H), 3.74-3.62 (m, 7H), 3.55 (t, J=5.2 Hz, 1H), 2.88 (t, J=5.2 Hz, 1H),2.81 (t, J=5.2 Hz, 1H), 1.64 (s, 2H), 1.48 (s, 9H).

Preparation of Compound 2e

DIPEA (0.042 mL, 0.32 mmol) and PyBOP (126 mg, 0.24 mmol) were added toa stirred mixture of compound 1i (200 mg, 0.16 mmol) and compound 2d (51mg, 0.19 mmol) in DMF (4 mL). After stirring at room temperature for 4hours under N₂, the reaction mixture was diluted with H₂O (100 mL) andextracted with EtOAc (2×100 mL). The combined organic layers were driedover anhydrous Na₂SO₄, filtered and concentrated. The resulting residuewas purified by column chromatography to yield the compound 2e (142 mg,60%). EI-MS m/z: [M+H]⁺ 1474.7.

Preparation of Compound 2f

To a solution of compound 2e (142 mg, 0.096 mmol) in MeOH (2 mL) wasadded LiOH monohydrate (36 mg, 0.86 mmol) in H₂O (2 mL) at −20° C. Afterstirred at 0° C. for 1 hour, the reaction mixture was diluted with H₂O/2N aq. HCl solution (50 mL/2 mL) and extracted with CHCl₃ (2×100 mL). Thecombined organic layers were dried over anhydrous Na₂SO₄, filtered andconcentrated to yield the crude compound 2f (128 mg), which was usedwithout further purification. EI-MS m/z: [M+H]⁺ 1334.5.

Preparation of Compound 2g

To a solution of crude compound 2f (105 mg, 0.08 mmol) in DCM (3 mL) HCl(4 M in 1,4-dioxane, 1 mL) was added at 0° C. After 1 hour, the solventand excess HCl were removed by N₂ flow and then the residue was purifiedby HPLC, which produced the compound 2g (47 mg, 46%) as white solid.EI-MS m/z: [M+H]⁺ 1234.4.

Example 4. Preparation of Compound 2h

Compound 2h was prepared from compound 1j and compound 2d by a similarmethod of preparing compound 2g in Example 3. EI-MS m/z: [M+H]⁺ 1248.9.

Example 5. Preparation of Compound 3f

Preparation of Compound 3a

A mixture of hexaethylene glycol (1.0 g, 3.54 mmol), Ag₂O (1.23 g, 5.31mmol) and KI (117 mg, 0.71 mmol) in DCM (10 mL) was sonicated for 15min. The suspension was cooled to −30° C. and a solution ofp-toluenesulfonyl chloride (688 mg, 3.61 mmol) in DCM (13 mL) was addeddropwise. The mixture was then gradually warmed up to 0° C. and kept for15 minutes at this temperature. Then the reaction mixture was dried overanhydrous MgSO₄, filtered and concentrated to produce the syrupyresidue. Then, the syrupy residue was purified by column chromatography(EtOAc to EtOAc/MeOH 10/1). The pure fractions were evaporated in vacuoto yield the compound 3a (1.18 g, 77%). ¹H-NMR (400 MHz, CDCl₃) δ 7.80(d, J=8.4 Hz, 2H), 7.34 (d, J=8.4 Hz, 2H), 4.16 (m, 2H), 3.72-3.58 (m,22H), 2.97 (br, 1H), 2.45 (s, 3H).

Preparation of Compound 3b

Compound 3a (1.18 g, 2.71 mmol) and NaN₃ (264 mg, 4.07 mmol) weredissolved in DMF (3 mL). And then the reaction mixture was heated at100° C. After 15 hours at 100° C., the reaction mixture was filtered andconcentrated. The residue was purified by column chromatography (EtOActo EtOAc/MeOH 10/1) to yield the compound 3b (728 mg, 87%). ¹H-NMR (400MHz, CDCl₃) δ 3.75-3.70 (m, 2H), 3.69-3.63 (m, 18H), 3.62-3.60 (m, 2H),3.39 (d, J=5.2 Hz, 2H), 3.07 (br, 1H).

Preparation of Compound 3c

To a stirred solution of compound 3b (728 mg, 2.36 mmol) in THF (10 mL)at 0° C. were added triethylamine (0.73 mL, 5.21 mmol) andmethanesulfonic anhydride (619 mg, 3.55 mmol). After 2 hours, LiBr (1.03g, 11.8 mmol) was added to a stirred solution and the resulting reactionmixture was refluxed for 5 hours. After cooling to room temperature, thereaction mixture was concentrated under reduced pressure. The residuewas purified by column chromatography (EtOAc to EtOAc/MeOH 10/1) toyield the compound 3c (810 mg, 92%). ¹H-NMR (400 MHz, CDCl₃) δ 3.81 (t,J=6.4 Hz, 2H), 3.69-3.65 (m, 18H), 3.47 (t, J=6.4 Hz, 2H), 3.39 (t,J=5.2 Hz, 2H).

Preparation of Compound 3d

NaH (60% in oil, 564 mg, 12.9 mmol) was added to a stirred mixture ofcompound 3c (3.42 g, 9.24 mmol) and N,N-diBoc-hydroxylamine (2.80 g,12.0 mmol, synthesized by the procedures in PCT publication No.WO2004/018466A2, hereby incorporated by reference) in DMF (20 mL) at 0°C. The reaction mixture was warmed to room temperature and kept for 2hours at this temperature. The solvent was evaporated under reducedpressure and the residue was purified by column chromatography(EtOAc/Hex 1/20 to 1/5), which produced the compound 3d (3.51 g, 73%).¹H-NMR (400 MHz, CDCl₃) δ 4.08 (t, J=4.8 Hz, 2H), 3.73 (t, J=4.8 Hz,2H), 3.69-3.62 (m, 18H), 3.39 (t, J=5.6 Hz, 2H), 1.53 (s, 18H).

Preparation of Compound 3e

To a stirred mixture of compound 3d (123 mg, 0.23 mmol), and Pd/C (10wt. %, 25 mg) in MeOH (5 mL) at 0° C. was added HCl (4 N in 1,4-dioxane,0.05 mL, 0.21 mmol). After stirring at room temperature for 5 hoursunder hydrogen, the reaction mixture was filtered through a celite padand washed with MeOH (100 mL). The filtrate was concentrated to producethe compound 3e (118 mg, 95%) as colorless oil, which was used withoutfurther purification. ¹H-NMR (400 MHz, DMSO-d₆) δ 3.98 (t, J=4.4 Hz,2H), 3.61-3.51 (m, 22H), 2.95 (br, 3H), 1.46 (s, 18H). EI-MS m/z: [M+H]⁺497.6.

Preparation of Compound 3f

Compound 3f was prepared from compound 1i and compound 3e by a similarmethod of preparing compound 2g in Example 3. EI-MS m/z: [M+H]⁺ 1366.6,[M+Na]⁺ 1389.6.

Example 6. Preparation of Compound 3g

Compound 3g was prepared from compound 1j and compound 3e by a similarmethod of preparing compound 2g in Example 3. EI-MS m/z: [M+H]⁺ 1380.6,[M+Na]⁺ 1403.6.

Example 7. Preparation of Compound 4f

Preparation of Compound 4a

To a stirred solution of dodecaethylene glycol (1.8 g, 3.2 mmol) in DCM(18 mL) was added p-toluenesulfonyl chloride (656 mg, 3.4 mmol), Ag₂O(1.13 g, 4.9 mmol) and KI (108 mg, 0.65 mmol). After stirring at roomtemperature for 30 minutes, the reaction mixture was filtered through acelite pad and washed with DCM (50 mL). The filtrate was concentrated.The resulting residue was purified by column chromatography to producethe compound 4a (490 mg, 21%) as light yellowish oil. ¹H-NMR (400 MHz,CDCl₃) δ 7.81 (d, 2H), 7.35 (d, 2H), 4.16 (t, 2H), 3.72-3.58 (m, 46H),2.82 (br s, 1H), 2.45 (s, 3H).

Preparation of Compound 4b

Compound 4a (490 mg, 0.69 mmol) and NaN₃ (68 mg, 1.04 mmol) weredissolved in DMF (16 mL) and the reaction mixture was heated at 100° C.for 3 hours. The reaction mixture was filtered and concentrated. Thecrude product was purified by column chromatography to yield thecompound 4b (267 mg, 67%). ¹H-NMR (400 MHz, CDCl₃) δ 3.72-3.60 (m, 46H),3.39 (t, 2H), 2.84 (t, 1H), 3.40 (m, 2H).

Preparation of Compound 4c

To a stirred solution of compound 4b (265 mg, 0.46 mmol) in THF (10 mL)at 0° C. were added 4-methylmorpholine (0.066 mL, 0.60 mmol) andmethanesulfonic anhydride (121 mg, 0.69 mmol). After 2 hours, LiBr (120mg, 1.38 mmol) was added to a stirred solution and the resultingreaction mixture was refluxed for 6 hours. After cooling to roomtemperature, the reaction mixture was concentrated under reducedpressure. The residue was purified by column chromatography (EtOAc toEtOAc/MeOH 10/1) to yield the compound 4c (178 mg, 60%). ¹H-NMR (400MHz, CDCl₃) δ 3.81 (t, 2H), 3.65 (m, 42H), 3.47 (t, 2H), 3.39 (t, 2H).

Preparation of Compound 4d

NaH (60% in oil, 14 mg, 0.33 mmol) was added to a stirred mixture ofcompound 4c (175 mg, 0.27 mmol), and N-Boc-hydroxylamine (47 mg, 0.35mmol) in DMF (5 mL) at 0° C. The reaction mixture was warmed up to roomtemperature and kept for 12 hours at this temperature. The solvent wasevaporated under reduced pressure and the residue was purified by columnchromatography (MeOH/CHCl₃ 1/20 to 1.5/20), which produced the compound4d (148 mg, 78%). ¹H-NMR (400 MHz, CDCl₃) δ 4.00 (t, 2H), 3.66 (m, 44H),3.39 (t, 2H), 1.47 (d, 9H).

Preparation of Compound 4e

To a stirred mixture of compound 4d (148 mg, 0.21 mmol), and Pd/C (10wt. %, 28 mg) in MeOH (5 mL) at 0° C. was added HCl (4 N in 1,4-dioxane,0.053 mL, 0.21 mmol). After stirring at room temperature for 30 minutesunder hydrogen, the reaction mixture was filtered through a celite padand washed with MeOH (30 mL). The filtrate was concentrated to producethe compound 4e (142 mg, 96%) as colorless oil, which was used withoutfurther purification. ¹H-NMR (400 MHz, DMSO-d₆) δ 4.00 (t, 2H), 3.92 (t,2H), 3.76-3.64 (m, 42H), 3.18 (t, 2H) 1.47 (s, 9H).

Preparation of Compound 4f

Compound 4f was prepared from compound 1i and compound 4e by a similarmethod of preparing compound 2g in Example 3. EI-MS m/z: [M+H]⁺ 1631.9.

Example 8. Preparation of Compound 4g

Compound 4g was prepared from compound 1j and compound 4e by a similarmethod of preparing compound 2g in Example 3. EI-MS m/z: EI-MS m/z[M+H]⁺ 1645.3.

Example 9. Preparation of Compound 5e

Preparation of Compound 5a

To a solution of 2-aminoethanol (10 g, 164 mmol) in DCM (70 mL) wereadded triethylamine (3.9 mL, 28.1 mmol) and benzyl chloroformate (30 mL,213 mmol) in DCM (30 mL) at 0° C. under N₂. After 24 hours, the reactionmixture was concentrated. The resulting residue was diluted with H₂O (50mL) and extracted with EtOAc (3×100 mL). The organic layers werecombined, dried over anhydrous MgSO₄, filtered and concentrated. Thecrude product was purified by column chromatography to produce thecompound 5a (17 g, 53%). ¹H-NMR (400 MHz, CDCl₃) δ 7.40-7.27 (m, 5H),5.11 (s, 2H), 3.72 (s, 2H), 3.56 (s, 2H), 2.13 (br s, 1H).

Preparation of Compound 5b

To a solution of compound 5a (5.0 g, 25.6 mmol) in DCM (70 mL)triethylamine (3.9 mL, 28.1 mmol) were added DMAP (100 mg, 5.12 mmol)and p-toluenesulfonyl chloride (5.4 g, 28.1 mmol) in DCM (30 mL) at 0°C. under N₂. After 15 hours at 0° C., the reaction mixture was dilutedwith saturated aq. NH₄Cl (100 mL) and extracted with DCM (2×100 mL). Theorganic layers were combined, dried over anhydrous MgSO₄, filtered andconcentrated. The crude product was purified by column chromatography toproduce the compound 5b (8.29 g, 92%). ¹H-NMR (400 MHz, CDCl₃) δ 7.77(d, J=7.6 Hz, 2H), 7.40-7.28 (m, 7H), 5.07 (s, 3H), 4.09 (s, 2H), 3.45(s, 2H), 2.43 (s, 3H).

Preparation of Compound 5c

To a solution of compound 5b (2.0 g, 7.23 mmol) in THF (20 mL) was addedN,N-diBoc-hydroxylamine (1.7 g, 7.44 mmol) and NaH (300 mg, 6.86 mmol)at 0° C. under N₂. After stirring at room temperature for 17 hours, thereaction mixture was diluted with saturated aqueous NH₄Cl (50 mL) andextracted with EtOAc (3×50 mL). The organic layers were combined, driedover anhydrous MgSO₄, filtered and concentrated. The crude product waspurified by column chromatography to produce the compound 5c (375 mg,16%). ¹H-NMR (400 MHz, CDCl₃) δ 7.45-7.27 (m, 5H), 5.11 (s, 2H), 4.01(br s, 2H), 3.44 (d, J=4.8 Hz, 2H), 1.52 (s, 18H). EI-MS m/z: [M+H]⁺410.7.

Preparation of Compound 5d

To a solution of compound 5c (187 mg, 0.45 mmol) in MeOH (20 mL) Pd/C(10% wt. %, 20 mg) was added and then the reaction mixture was stirredat room temperature for 4 hours under hydrogen. The reaction mixture wasfiltered through a celite pad and washed with MeOH (20 mL). The filtratewas concentrated to produce the compound 5d (120 mg) as colorless oil,which was used without further purification.

Preparation of Compound 5e

Compound 5e was prepared from compound 1i and compound 5d by a similarmethod of preparing compound 2g in Example 3. EI-MS m/z: [M+H]⁺ 1146.4.

Example 10. Preparation of Compound 5f

Compound 5f was prepared from compound 1j and compound 5d by a similarmethod of preparing compound 2g in Example 3. EI-MS m/z: [M+H]⁺ 1160.3.

Example 11. Preparation of Compound 6e

Preparation of Compound 6a

DIPEA (1.2 mL, 9.96 mmol) and HBTU (1.69 g, 6.22 mmol) were added to astirred mixture of Z-Asp(OMe)-OH (500 mg, 1.78 mmol) and compound 2d(642 mg, 2.98 mmol) in DMF (5 mL). The reaction mixture was stirred atroom temperature for 22 hours under N₂. The reaction mixture was dilutedwith H₂O (100 mL) and extracted with EtOAc (2×100 mL). The combinedorganic layers were dried over anhydrous Na₂SO₄, filtered andconcentrated. The resulting residue was purified by columnchromatography to yield the compound 6a (368 mg, 40%). ¹H-NMR (400 MHz,CDCl₃) δ 7.85-7.70 (m, 1H), 7.45-7.28 (m, 5H), 7.04 (s, 1H), 6.02 (d,J=8.4 Hz, 1H), 5.11 (s, 2H), 4.65-4.50 (m, 1H), 4.00 (d, J=3.6 Hz, 2H),3.72-3.30 (m, 10H), 2.80 (dd, J=5.6 Hz, 2H), 1.46 (s, 9H).

Preparation of Compound 6b

To a stirred mixture of compound 6a (150 mg, 0.28 mmol) and Pd/C (10 wt.%, 20 mg) in MeOH (5 mL) at 0° C. was added HCl (4 N in 1,4-dioxane,0.07 mL, 0.28 mmol). After stirring at room temperature for 2 hoursunder hydrogen, the reaction mixture was filtered through a celite padand washed with MeOH (20 mL). The filtrate was concentrated to producethe compound 6b (169 mg) as colorless oil, which was used withoutfurther purification. EI-MS m/z: [M+H]⁺ 393.7.

Preparation of Compound 6c

DIPEA (0.022 mL, 0.12 mmol) and HBTU (20 mg, 0.05 mmol) were added to astirred mixture of compound 1i (50 mg, 0.04 mmol) and compound 6b (22mg, 0.05 mmol) in DMF (1 mL). The reaction mixture was stirred at roomtemperature for 14 hours under N₂. Then, the reaction mixture wasdiluted with H₂O (10 mL) and extracted with EtOAc (2×20 mL). Thecombined organic layers were dried over anhydrous Na₂SO₄, filtered andconcentrated. The resulting residue was purified by columnchromatography to yield the compound 6c (38 mg, 60%). EI-MS m/z: [M+H]⁺1604.5.

Preparation of Compound 6d

To a solution of compound 6c (38 mg, 0.023 mmol) in MeOH (1 mL) wasadded LiOH monohydrate (5 mg, 0.118 mmol) in H₂O (1 mL) at 0° C. After 2hours at 0° C., the pH of the solution was adjusted with AcOH to 4-5 andconcentrated under reduced pressure. The residue was dissolved in DMSO(1.5 mL) and purified by HPLC to produce the compound 6d (26 mg, 78%).

EI-MS m/z: [M+H]⁺ 1450.5.

Preparation of Compound 6e

TFA (0.3 mL) was added to a stirred solution of compound 6d (26 mg,0.018 mmol) in DCM (1.5 mL). After stirring at 0° C. for 2 hours, thesolvent and excess TFA were removed by N₂ flow. Then the residue wasdissolved in DMSO (1 mL) and purified by HPLC. Pure fractions with thesame retention time were combined and lyophilized to produce thecompound 6e (19.5 mg, 80%) as white solid. EI-MS m/z: [M+H]⁺ 1350.6.

Example 12. Preparation of Compound 7e

Preparation of Compound 7a

NaH (60 wt. %, 500 mg, 12.49 mmol) was added to a stirred mixture ofcompound 4c (6.10 g, 9.61 mmol) and N,N-diBoc-hydroxylamine (2.69 g,11.53 mmol) in DMF (90 mL) at 0° C. The reaction mixture was heated upto room temperature and kept for 12 hours at this temperature. Thereaction mixture was evaporated under reduced pressure and the resultingresidue was purified by column chromatography. Pure fractions wereevaporated in vacuo to yield the compound 7a (5.70 g, 75%). ¹H-NMR (400MHz, CDCl₃) δ 4.05 (t, 2H), 3.71 (t, 2H), 3.64 (m, 42H), 3.37 (t, 2H),1.51 (d, 18H).

Preparation of Compound 7b

To a stirred mixture of compound 7a (5.70 g, 7.21 mmol), and Pd/C (10wt. %, 570 mg) in MeOH (100 mL) at 0° C. was added HCl (4 N in1,4-dioxane, 1.9 mL, 7.2 mmol). After stirring at room temperature for30 minutes under hydrogen, the reaction mixture was filtered through acelite pad and washed with MeOH (30 mL). The filtrate was concentratedto produce the compound 7b (5.10 g, 87%) as colorless oil, which wasused without further purification. ¹H-NMR (400 MHz, DMSO-d₆) δ 4.21 (t,2H), 4.07 (s, 2H), 3.95-3.78 (m, 42H), 3.32 (s, 2H) 1.63 (s, 18H).

Preparation of Compound 7c

DIPEA (0.25 mL, 1.42 mmol) and HBTU (337 g, 0.89 mmol) were added to astirred mixture of Z-Asp(OMe)-OH (100 mg, 0.36 mmol) and compound 7b(340 mg, 0.43 mmol) in DMF (10 mL). The reaction mixture was stirred atroom temperature for 20 hours under N₂. Then, the reaction mixture wasdiluted with H₂O (100 mL) and extracted with EtOAc (2×100 mL). Thecombined organic layers were dried over anhydrous Na₂SO₄, filtered andconcentrated. The resulting residue was purified by columnchromatography to yield the compound 7c (123 mg, 58%). EI-MS m/z: [M+H]⁺1024.2.

Preparation of Compound 7d

To a stirred mixture of compound 7c (120 mg, 0.12 mmol) and Pd/C (10 wt.%, 20 mg) in MeOH (5 mL) at 0° C. was added HCl (4 N in 1,4-dioxane,0.03 mL, 0.12 mmol). After stirring at room temperature for 2 hoursunder hydrogen, the reaction mixture was filtered through a celite padand washed with MeOH (20 mL). The filtrate was concentrated to producethe compound 7d (120 mg) as colorless oil, which was used withoutfurther purification.

Preparation of Compound 7e

Compound 7e was prepared from compound 1i and compound 7d by a similarmethod of preparing compound 6e in Example 11. EI-MS m/z: [M+H]⁺ 1747.1.

Example 13. Preparation of Compound 8f

Preparation of Compound 8a

To a solution of 2-(2-(2-chloroethoxy)ethoxy)ethanol (5.0 g, 29.6 mmol)in acetone (30 mL) was added NaI (13.3 g, 88.9 mmol). The reactionmixture was refluxed for 12 hours. After the reaction was completed, thereaction mixture was filtered and concentrated. The crude product waspurified by column chromatography to produce the compound 8a (7.0 g,91%). ¹H-NMR (400 MHz, CDCl₃) δ 3.80-3.73 (m, 4H), 3.72-3.65 (m, 4H),3.63-3.61 (m, 2H), 3.27 (t, J=6.4 Hz, 2H).

Preparation of Compound 8b

NaH (500 mg, 12.49 mmol) was added to a stirred mixture of compound 8a(2.0 g, 7.69 mmol), and N,N-diBoc-hydroxylamine (2.33 g, 10.00 mmol) inDMF (20 mL) at 0° C. under N₂. After stirring at room temperature for 17hours, the reaction mixture was diluted with saturated aq. NH₄Cl (50 mL)and extracted with EtOAc (3×50 mL). The organic layers were combined,dried over anhydrous MgSO₄, filtered and concentrated. The crude productwas purified by column chromatography to produce the compound 8b (1.54g, 54%). ¹H-NMR (400 MHz, CDCl₃) δ 7.45-7.27 (m, 5H), 5.11 (s, 2H), 4.01(br s, 2H), 3.44 (d, J=4.8 Hz, 2H), 1.52 (s, 18H). EI-MS m/z: [M+H]⁺410.7.

Preparation of Compound 8c

To a stirred solution of the compound 8b (123 mg, 0.242 mmol) in DMSO (2mL) and DCM (2 mL) were added SO₃.pyridine complex (116 mg, 0.726 mmol)and triethylamine (0.17 mL, 1.21 mmol) at 0° C. under N₂. After 1 hour,the reaction mixture was diluted with saturated aq. NH₄Cl (10 mL) andextracted with DCM (2×10 mL). The organic layers were combined, driedover anhydrous MgSO₄, and filtered. Concentration under reduced pressureprovided the compound 8c (88 mg), which was used without furtherpurification. ¹H-NMR (400 MHz, CDCl₃) δ 9.74 (s, 1H), 4.19 (s, 2H),3.77-3.69 (m, 6H), 3.42 (m, 2H).

Preparation of Compound 8d

To a solution of β-glutamic acid (500 mg, 0.339 mmol) in MeOH (10 mL)was added thionyl chloride (0.148 mL, 2.04 mmol) at 0° C. under N₂.After 24 hours, the reaction mixture was concentrated to produce thecompound 8d (697 mg), which was used without further purification.¹H-NMR (400 MHz, CDCl₃) δ 7.40-7.27 (m, 5H), 5.11 (s, 2H), 3.72 (s, 2H),3.56 (s, 2H), 2.13 (br s, 1H).

Preparation of Compound 8e

To a solution of compound 8d (34 mg, 0.16 mmol) and compound 8c (88 mg,0.24 mmol) in MeOH (5 mL) was added NaCNBH₃ (10 mg, 0.16 mmol) at roomtemperature under N₂. After 3 hours, the reaction mixture was filteredand concentrated. The crude product was purified by columnchromatography to produce the compound 8e (53 mg, 63%). ¹H-NMR (400 MHz,CDCl₃) δ 7.45-7.25 (m, 10H), 5.60 (br s, 2H), 5.03 (s, 4H), 3.80-3.25(m, 20H), 2.81 (s, 4H).

Preparation of Compound 8f

Compound 8f was prepared from compound 1i and compound 8e by a similarmethod of preparing compound 6e in Example 11. EI-MS m/z: [M+H]⁺ 1365.0.

Example 14. Preparation of Compound 9j

Preparation of Compound 9a

To a solution of hexaethylene glycol (10.48 g, 37.12 mmol) in DCM (400mL) was added imidazole (3.20 g, 44.54 mmol) at 0° C. under N₂. After 5minutes, the reaction mixture was added dropwise to the solution ofTBSCl (5.60 g, 37.12 mmol) in DCM (50 mL) at the same temperature underN₂ atmosphere. The reaction mixture was stirred at 0° C. and warmed toroom temperature for 21 hours under N₂. After the reaction wascompleted, the reaction mixture was diluted with water (200 mL) andextracted with DCM (2×100 mL). The organic layers were dried overanhydrous Na₂SO₄ and concentrated under reduced pressure. The resultingresidue was purified by column chromatography. The pure fractions wereevaporated in vacuo to yield the compound 9a (6.70 g, 46%). ¹H-NMR (400MHz, CDCl₃) δ 3.77-3.71 (m, 4H), 3.66-3.60 (m, 18H), 3.56-3.54 (t, 2H),0.89 (s, 9H), 0.06 (s, 6H).

Preparation of Compound 9b

To a solution of compound 9a (3.32 g, 8.37 mmol) in dry THF (40 mL) wasadded NaH (55% in oil, 438 mg, 10.05 mmol) at 0° C. under N₂. After 30minutes, MeI (0.78 mL, 12.56 mmol) was added to the reaction mixture atthe same temperature under N₂. The reaction mixture was stirred andwarmed to room temperature for 18 hours under N₂. After the reaction wascompleted, quenched with H₂O (10 mL) and extracted with EA (3×10 mL).The organic layers were combined, washed with saturated aq. NH₄Cl (5 mL)and brine (10 mL), dried over anhydrous Na₂SO₄ and evaporated underreduced pressure. The resulting residue was purified by columnchromatography. Pure fractions were evaporated in vacuo to yield thecompound 9b (3.16 g, 92%). ¹H-NMR (400 MHz, CDCl₃) δ 3.78-3.75 (t, 2H),3.65 (s, 20H), 3.57-3.54 (t, 4H), 3.38 (s, 3H), 0.89 (s, 9H), 0.06 (s,6H).

Preparation of Compound 9c

To a solution of compound 9b (3.16 g, 7.69 mmol) in acetone (100 mL) wasadded Jones reagent (10 mL) at 0° C. under N₂. The reaction mixture wasstirred and warmed to room temperature for 17 hours under N₂. After thereaction was completed, the reaction mixture was filtered and evaporatedunder reduced pressure. The residue was diluted with H₂O (100 mL) andextracted with CHCl₃ (3×50 mL). The organic layers were combined, driedover anhydrous Na₂SO₄ and evaporated under reduced pressure. Theresulting crude compound 9c (2.28 g, 95%) was used without furtherpurification. ¹H-NMR (400 MHz, CDCl₃) δ 4.16 (s, 2H), 3.76-3.75 (t, 2H),3.69-3.67 (m, 16H), 3.57-3.55 (t, 2H), 3.38 (s, 3H).

Preparation of Compound 9d

DIPEA (3.8 mL, 22.03 mmol), HOBt (1.29 g, 9.55 mmol) and EDC.HCl (1.83g, 9.55 mmol) were added to a stirred mixture of compound 9c (2.28 g,7.34 mmol) and H-Lys(Z)—OMe hydrochloride (2.91 g, 8.81 mmol) in DMF (30mL). After stirring at room temperature for 14 hours under N₂, thereaction mixture was concentrated. Purification by column chromatographygave the compound 9d (1.23 g, 72%). ¹H-NMR (400 MHz, CDCl₃) δ 7.38-7.31(m, 5H), 5.10 (s, 2H), 5.00 (s, 1H) 4.68-4.62 (m, 1H), 4.03 (s, 2H),3.75 (s, 3H), 3.68-3.64 (m, 16H), 3.56 (t, 2H), 3.39 (s, 3H), 3.20 (m,2H), 1.89 (m, 1H), 1.74 (m, 1H), 1.55 (m, 1H), 1.40 (m, 1H). EI-MS m/z:[M+H]⁺ 586.8, [M+Na]⁺ 608.9.

Preparation of Compound 9e

To a solution of compound 9d (2.16 g, 3.68 mmol) in THF/MeOH/H₂O (18mL/6 mL/6 mL) was added LiOH monohydrate (307 mg, 7.31 mmol) at 0° C.under N₂. The reaction mixture was stirred for 1 hour at roomtemperature. Then the pH of the solution was adjusted to 2-3 with 1 Naq. HCl. The reaction mixture was poured into H₂O (20 mL) and extractedwith DCM (3×50 mL). The organic layers were combined, dried over Na₂SO₄.Filtration and concentration produced the compound 9e (2.28 g, 99%),which was used without further purification. ¹H-NMR (400 MHz, CDCl₃) δ7.34-7.30 (m, 5H), 5.08 (s, 2H), 4.66-4.60 (q, 1H), 4.01 (s, 2H),3.67-3.55 (m, 18H), 3.37 (s, 3H), 3.20 (m, 2H), 1.87 (m, 1H), 1.72 (m,1H), 1.53 (m, 1H), 1.38 (m, 1H).

Preparation of Compound 9f

DIPEA (0.45 mL, 2.63 mmol), HOBt (154 mg, 0.11 mmol) and EDC.HCl (218mg, 0.11 mmol) were added to a stirred mixture of compound 9e (502 mg,0.88 mmol) and compound 7b (700 mg, 0.88 mmol) in DMF (8 mL). Afterstirring at room temperature for 14 hours under N₂, the reaction mixturewas poured into H₂O (20 mL) and extracted with EtOAc (3×20 mL). Thecombined organic layers were washed with aq. NaHCO₃ (20 mL) and brine(20 mL) and dried over anhydrous Na₂SO₄. After filtration andconcentration under reduced pressure, the resulting residue was purifiedby column chromatography to yield the compound 9f (499 mg, 43%). ¹H-NMR(400 MHz, CDCl₃) δ 7.35-7.30 (m, 5H), 6.83 (s, 1H), 5.15 (s, 1H), 5.08(s, 2H), 4.43 (q, 1H) 4.07 (t, 1H), 3.65-3.60 (m, 54H), 3.55-3.53 (m,4H), 3.37 (s, 3H), 3.16 (m, 2H), 1.85 (m, 1H), 1.53-1.52 (d, 19H), 1.38(m, 2H). EI-MS m/z: [M+H]⁺ 1337.5.

Preparation of Compound 9g

To a stirred mixture of compound 9f (499 mg, 0.37 mmol) and Pd/C (10 wt.%, 50 mg) in MeOH (20 mL) at 0° C. was added HCl (4 N in 1,4-dioxane,0.1 mL, 0.37 mmol). After stirring at room temperature for 90 minutesunder hydrogen, the reaction mixture was filtered through a celite padand washed with MeOH (10 mL). The filtrate was concentrated to producethe compound 9g (458 mg, 98%) as colorless oil, which was used withoutfurther purification. EI-MS m/z: [M+H]⁺ 1218.6.

Preparation of Compound 9h

DIPEA (0.019 mL, 0.11 mmol) and HBTU (18 mg, 0.05 mmol) were added to astirred mixture of compound 1i (45 mg, 0.04 mmol) and compound 9g (57mg, 0.05 mmol) in DMF (0.5 mL). The reaction mixture was stirred at roomtemperature for 14 hours under N₂. The reaction mixture was diluted withH₂O/DMSO (1.5 mL/1.5 mL) and purified by HPLC, which produced compound9h (65 mg, 57%). EI-MS m/z: 1/2[M+H]⁺ 1181.7.

Preparation of Compound 9i

To a solution of compound 9h (65 mg, 0.03 mmol) in MeOH (1.5 mL) wasadded LiOH monohydrate (10 mg, 0.24 mmol) in H₂O (1.5 mL) at 0° C. After1 hour at 0° C., the pH of the solution was adjusted with AcOH to 4-5and concentrated under reduced pressure. Then the reaction mixture wasdissolved in H₂O/DMSO (1.5 mL/1.5 mL) and purified by HPLC, whichproduced compound 9i (45 mg, 55%). EI-MS m/z: 1/2[M+Na]⁺ 1098.7.

Preparation of Compound 9j

TFA (0.2 mL) was added to a stirred solution of compound 9i (45 mg, 0.02mmol) in DCM (1 mL). After stirring at 0° C. for 30 minutes, the solventand excess TFA were removed by N₂ flow. Then the residue was dissolvedin H₂O/DMSO (1 mL/1 mL) and purified by HPLC. Pure fractions with thesame retention time were combined and lyophilized to produce thecompound 9j (14 mg, 32%) as white solid. EI-MS m/z: 1/2[M+H]⁺1026.3.

Example 15. Preparation of Compound 10c

Preparation of Compound 10a

DIPEA (0.03 mL, 0.17 mmol), HOBt (10 mg, 0.075 mmol) and EDC.HCl (14 mg,0.075 mmol) were added to a stirred mixture of compound 9e (33 mg, 0.058mmol) and compound 7d (54 mg, 0.058 mmol) in DMF (3 mL). After stirringat room temperature for 14 hours under N₂, the reaction mixture waspoured into H₂O (10 mL) and extracted with EtOAc (3×10 mL). The combinedorganic layers were washed with 1 N aq. HCl (8 mL), saturated aq. NaHCO₃(8 mL) and brine (8 mL), and dried over anhydrous Na₂SO₄. Afterfiltration and concentration, the residue was purified by columnchromatography, which produced the compound 10a (61 mg, 73%). EI-MS m/z:[M+H]⁺ 1445.0, [M+H-Boc]⁺ 1344.9.

Preparation of Compound 10b

To a stirred mixture of compound 10a (60 mg, 0.04 mmol), and Pd/C (10wt. %, 30 mg) in MeOH (10 mL) at 0° C. was added HCl (4 N in1,4-dioxane, 0.01 mL, 0.01 mmol). After stirring at room temperature for3 hours under hydrogen, the reaction mixture was filtered through acelite pad and washed with MeOH (40 mL). The filtrate was concentratedto produce the compound 10b (56 mg, 100%) as colorless oil, which wasused without further purification. EI-MS m/z: [M+H]⁺ 1311.0, [M+Na+]⁺1332.9.

Preparation of Compound 10c

Compound 10c was prepared from compound 1i and compound 10b by a similarmethod of preparing compound 9j in Example 14. EI-MS m/z: 1/2[M+H]⁺1083.8.

Example 16. Preparation of Compound 10d

Compound 10d was prepared from compound 1j and compound 10b by a similarmethod of preparing compound 9j in Example 14. EI-MS m/z: [M+H]⁺ 2181.3,1/2[M+H]⁺ 1091.3.

Example 17. Preparation of Compound 11j

Preparation of Compound 11a

To a solution of compound 3a (8.0 g, 18.3 mmol) in THF (50 mL) was addedLiBr (7.9 g, 91.6 mmol) at room temperature. After stirring for 17 hoursunder reflux, the reaction mixture was filtered and concentrated. Thecrude product was purified by column chromatography to produce thecompound 11a (3.2 g, 50%). ¹H-NMR (400 MHz, CDCl₃) δ 3.95-3.50 (m, 24H).

Preparation of Compound 11b

To a solution of compound 11a (3.2 g, 12.3 mmol) in acetone (20 mL) at0° C. was added Jones reagent (20 mL). After 15 hours at 0° C., thereaction mixture was filtered and concentrated. The residue was dilutedwith H₂O (50 mL) and extracted with EtOAc (2×100 mL). The organic layerswere combined, dried over anhydrous MgSO₄, filtered and concentrated.The crude product was purified by column chromatography to produce thecompound 11b (3.2 g, 72%). ¹H-NMR (400 MHz, CDCl₃) δ 4.16 (s, 2H),3.95-3.30 (m, 20H).

Preparation of Compound 11e

To a solution of compound 11b (3.2 g, 8.90 mmol) in MeOH (30 mL) wasadded oxalyl chloride (1.15 mL, 13.3 mmol) at 0° C. under N₂. After 16hours, the reaction mixture was concentrated and purified by columnchromatography, which produced the compound 11c (2.7 g, 81%). ¹H-NMR(400 MHz, CDCl₃) δ 4.17 (s, 2H), 3.80-3.60 (m, 21H), 3.47 (t, J=6.4 Hz,2H).

Preparation of Compound 11d

Compound 11e (1.0 g, 2.67 mmol) and NaN₃ (261 mg, 4.01 mmol) weredissolved in DMF (3 mL). The reaction mixture was heated at 100° C. for5 hours. After the reaction was completed, the reaction mixture wasfiltered and concentrated. The residue was purified by columnchromatography (EtOAc to EtOAc/MeOH 10/1), which produced the compound11d (854 mg, 95%). ¹H-NMR (400 MHz, CDCl₃) δ 4.17 (s, 2H), 3.76-3.64 (m,21H), 3.39 (t, J=5.2 Hz, 2H).

Preparation of Compound 11e

To a stirred solution of compound 11d (854 mg, 2.54 mmol) in MeOH (25mL) at 0° C. was added 2 M aq. NaOH (6.3 mL, 12.64 mmol). The reactionmixture was stirred at room temperature for 3 hours. The solution wasthen concentrated under reduced pressure. The resulting suspension wasacidified with aqueous 2 N HCl while cooling at 0° C. The residue wasextracted by CHCl₃ (8×50 mL). The organic layers were combined, driedover Na₂SO₄ and concentrated to produce the compound 11e (783 mg, 96%).¹H-NMR (400 MHz, CDCl₃) δ 4.16 (s, 2H), 3.76-3.65 (m, 18H), 3.40 (t,J=5.2 Hz, 2H).

Preparation of Compound 11f

DIPEA (0.47 mL, 2.72 mmol), HOBt (160 mg, 1.18 mmol) and EDC.HCl (226mg, 1.18 mmol) were added to a stirred mixture of compound 9e (520 mg,0.91 mmol) and compound 2d (270 mg, 0.91 mmol) in DMF (5 mL). Afterstirring at room temperature for 14 hours under N₂, the reaction mixturewas poured into H₂O (20 mL) and extracted with EtOAc (3×30 mL). Thecombined organic layers were washed with 1 N aq. HCl (15 mL), saturatedaq. NaHCO₃ (15 mL) and brine (15 mL), and dried over anhydrous Na₂SO₄.After filtration and concentration, the residue was purified by columnchromatography, which produced the compound 11f (631 mg, 85%).

EI-MS m/z: [M+H]⁺ 819.1, [M+H-Boc]⁺ 719.1 [M+Na+]⁺ 841.1.

Preparation of Compound 11g

To a stirred mixture of compound 11f (300 mg, 0.36 mmol), and Pd/C (10wt. %, 70 mg) in MeOH (20 mL) at 0° C. was added HCl (4 N in1,4-dioxane, 0.08 mL, 0.08 mmol). After stirring at room temperature for3 hours under hydrogen, the reaction mixture was filtered through acelite pad and washed with MeOH (40 mL). The filtrate was concentratedto produce the compound 11g (200 mg, 99%) as colorless oil, which wasused without further purification. EI-MS m/z: [M+H]⁺ 685.1, [M+Na]⁺707.1.

Preparation of Compound 11h

DIPEA (0.024 mL, 0.41 mmol), HOBt (24 mg, 0.18 mmol) and EDC.HCl (34 mg,0.18 mmol) were added to a stirred mixture of compound 11g (100 mg, 0.14mmol) and compound 11e (44 mg, 0.14 mmol) in DMF (5 mL). After stirringat room temperature for 14 hours under N₂, the reaction mixture waspoured into H₂O (10 mL) and extracted with DCM (3×10 mL). The combinedorganic layers were dried over anhydrous Na₂SO₄. After filtration andconcentration, the residue was purified by column chromatography, whichproduced the compound 11h (73 mg, 53%). EI-MS m/z: [M+H]⁺ 988.4,[M+Na-Boc]⁺ 888.2, [M+Na]⁺ 1010.4.

Preparation of Compound 11i

To a stirred mixture of compound 11h (73 mg, 0.07 mmol), and Pd/C (10wt. %, 10 mg) in MeOH (7 mL) at 0° C. was added HCl (4 N in 1,4-dioxane,0.018 mL, 0.018 mmol). After stirring at room temperature for 2 hoursunder hydrogen, the reaction mixture was filtered through a celite padand washed with MeOH (30 mL). The filtrate was concentrated to producethe compound 11i (72 mg, 99%) as colorless oil, which was used withoutfurther purification. EI-MS m/z: [M+H]⁺ 962.4, [M+Na]⁺ 984.4.

Preparation of Compound 11j

Compound 11j was prepared from compound 1i and compound 11i by a similarmethod of preparing compound 9j in Example 14. EI-MS m/z: [M+H]⁺ 1932.5.

Example 18. Preparation of Compound 11k

Compound 11k was prepared from compound 1j and compound 11i by a similarmethod of preparing compound 9j in Example 14. EI-MS m/z: [M+H]⁺ 1947.1.

Example 19. Preparation of Compound 12c

Preparation of Compound 12a

DIPEA (0.13 mL, 0.77 mmol) and HBTU (110 mg, 0.35 mmol) were added to astirred mixture of compound 9g (235 mg, 0.1929 mmol) and Z-Asp(OMe)-OH(54 mg, 0.212 mmol) in DMF (5 mL). After stirring at room temperaturefor 14 hours under N₂, the reaction mixture was poured into H₂O (20 mL)and extracted with EtOAc (3×20 mL). The combined organic layers werewashed with 1 N aq. HCl (7 mL), saturated aq. NaHCO₃ (7 mL) and brine (7mL), and dried over anhydrous Na₂SO₄. After filtration andconcentration, the residue was purified by column chromatography, whichproduced the compound 12a (260 mg, 93%). ¹H-NMR (400 MHz, CDCl₃) δ 8.07(t, 1H), 7.62 (t, 1H), 7.54-7.52 (m, 1H), 5.73 (s, 2H), 4.27-4.25 (q,1H), 3.96 (t, 2H), 3.88 (s, 2H), 3.82 (s, 2H), 3.58-3.48 (m, 52H),3.19-3.18 (m, 3H), 3.04-3.03 (m, 3H), 1.44 (s, 18H), 1.39-1.37 (m, 3H),1.21-1.19 (m, 3H). EI-MS m/z: [M+H−2Boc]⁺ 1031.6.

Preparation of Compound 12b

To a stirred mixture of compound 12a (260 mg, 0.179 mmol), and Pd/C (10wt. %, 72 mg) in MeOH (20 mL) at 0° C. was added HCl (4 N in1,4-dioxane, 0.040 mL, 0.179 mmol). After stirring at room temperaturefor 3 hours under hydrogen, the reaction mixture was filtered through acelite pad and washed with MeOH (40 mL). The filtrate was concentratedto produce the compound 12b (242 mg, 100%) as colorless oil, which wasused without further purification. EI-MS m/z: [M+H]⁺ 625.0, [M+H-Boc]⁺525.0, [M+H−2Boc]⁺ 424.9.

Preparation of Compound 12c

Compound 12c was prepared from compound 1i and compound 12b by a similarmethod of preparing compound 9j in Example 14. EI-MS m/z: 1/2[M+H]⁺1083.5.

Example 20. Preparation of Compound 12d

Compound 12d was prepared from compound 1j and compound 12b by a similarmethod of preparing compound 9j in Example 14. EI-MS m/z: 1/2[M+H]⁺1090.5.

Example 21. Preparation of Compound 13e

Preparation of Compound 13a

DIPEA (0.22 mL, 1.25 mmol) and HBTU (356 mg, 0.94 mmol) were added to astirred mixture of Z-Glu(OMe)-OH (222 mg, 0.75 mmol) and compound 7b(500 mg, 0.62 mmol) in DMF (5.0 mL). The reaction mixture was stirred atroom temperature for 14 hours under N₂. The reaction mixture was dilutedwith water (200 mL) and extracted with EA (3×100 mL). The organic layerswere dried over anhydrous MgSO₄, filtered and concentrated under reducedpressure. The resulting residue was purified by column chromatography toyield the compound 13a (370 mg, 57%). ¹H-NMR (400 MHz, CDCl₃) δ 7.34(br, 5H), 6.73 (br, 1H), 5.72 (d, J=7.6 Hz, 1H), 5.06 (br, 2H),4.28-4.18 (m, 1H), 4.07 (t, J=4.4 Hz, 2H), 3.76-3.71 (m, 2H), 3.70-3.50(m, 45H), 3.48-3.42 (m, 2H), 2.53-2.36 (m, 2H), 2.20-2.08 (m, 1H),2.00-1.88 (m, 1H), 1.53 (s, 18H). EI-MS m/z: [M+Na]⁺ 1061.2.

Preparation of Compound 13b

4N HCl in 1,4-dioxane (0.08 mL, 0.32 mmol) was added to a stirredmixture of the compound 13a (370 mg, 0.35 mmol), and Pd/C (38 mg) inMeOH (8 mL) at 0° C. After stirring at room temperature for 20 hoursunder hydrogen, the reaction mixture was filtered through a celite padand washed with MeOH (400 mL). The filtrate was concentrated, producingcompound 13b (301 mg, 90%) as yellow liquid, which was used withoutfurther purification. ¹H-NMR (400 MHz, CDCl₃) δ 8.41 (br, 1H), 8.09 (br,3H), 4.13 (br, 1H), 3.85-3.56 (m, 51H), 2.55 (br, 2H), 2.38-2.18 (m,2H), 1.53 (s, 18H). EI-MS m/z: [M+H]⁺ 905.0.

Preparation of Compound 13c

DIPEA (0.165 mL, 0.96 mmol) and HBTU (279 mg, 0.74 mmol) were added to astirred mixture compound 13b (300 mg, 0.32 mmol) and compound 9e (366mg, 0.64 mmol) in DMF (5.0 mL). The reaction mixture was stirred at roomtemperature for 14 hours under N₂. The reaction mixture was diluted withwater (200 mL) and extracted with EtOAc (3×100 mL). The organic layerswere dried over anhydrous MgSO₄, filtered and concentrated under reducedpressure. The resulting residue was purified by column chromatography toyield the compound 13c (290 mg, 62%). ¹H-NMR (400 MHz, CDCl₃) δ7.40-7.32 (m, 7H), 7.00 (br, 1H), 6.73 (br, 1H), 5.07 (br, 2H),4.44-4.36 (m, 2H), 4.07 (t, J=4.8 Hz, 2H), 4.02 (br, 2H), 3.73 (t, J=5.2Hz, 2H), 3.71-3.52 (m, 68H), 3.24-3.14 (m, 2H), 2.52-2.34 (m, 3H),2.18-2.06 (m, 2H), 1.98-1.82 (m, 4H), 1.76-1.64 (m, 3H), 1.53 (s, 18H).EI-MS m/z: [M+H]⁺ 1459.7.

Preparation of Compound 13d

Pd/C (21 mg) was added to a stirred mixture of compound 13c (290 mg,0.19 mmol) in MeOH (5 mL) at 0° C. After stirring at room temperaturefor 20 hours under hydrogen, the reaction mixture was filtered through acelite pad and washed with MeOH (400 mL). The filtrate was concentrated,producing compound 13c (247 mg, 94%) as yellow liquid, which was usedwithout further purification. ¹H-NMR (400 MHz, CDCl₃) δ 8.20 (d, J=8.4Hz, 1H), 7.74 (br, 1H), 7.30 (br, 1H), 4.66-4.48 (m, 2H), 4.07 (t, J=5.2Hz, 2H), 4.01 (br, 2H), 3.74-3.62 (m, 70H), 3.57-3.53 (m, 2H), 3.04-2.98(m, 2H), 2.24-2.15 (m, 2H), 2.14-2.06 (m, 2H), 1.99-1.86 (m, 4H),1.84-1.74 (m, 2H), 1.53 (s, 18H). EI-MS m/z: [M+H]⁺ 1325.5.

Preparation of Compound 13e

Compound 13e was prepared from compound 1i and compound 13d by a similarmethod of preparing compound 9j in Example 14. EI-MS m/z: [M+H]⁺ 2181.5.

Example 22. Preparation of Compound 13f

Compound 13f was prepared from compound 1j and compound 13d by a similarmethod of preparing compound 9j in Example 14. EI-MS m/z: [M+H]⁺ 2195.5.

Example 23. Preparation of Compound 14m

Preparation of Compound 14a

To a solution of 6-amino-1-hexanol (5.0 g, 42.6 mmol) in DCM (30 mL) wasadded di-tert-butyl dicarbonate (9.3 g, 42.6 mmol) at room temperature.After stirring for 18 hours, triethylamine (8.7 mL, 63.9 mmol) andt-butyldimethylsilyl chloride (7.7 g, 51.2 mmol) were added to thereaction mixture at 0° C. After 24 hours at room temperature, thereaction mixture diluted with saturated aq. NH₄Cl (200 mL). Theresulting mixture was extracted with EtOAc (100 mL). The organic layerwas washed with brine (100 mL) and dried over anhydrous MgSO₄, filteredand concentrated. The residue was purified by column chromatography toproduce the compound 14a (12 g, 84%). ¹H-NMR (400 MHz, CDCl₃) δ 4.50 (brs, 1H), 3.58 (t, J=6.8 Hz, 2H), 3.10 (d, J=6.4 Hz, 2H), 1.72-1.20 (m,17H), 0.88 (s, 9H), 0.04 (s, 6H).

Preparation of Compound 14b

To a solution of compound 14a (6.0 g, 18.1 mmol) in THF (30 mL) wereadded NaH (60% in oil, 2.4 g, 54.2 mmol) and methyl iodide (3.4 mL, 54.2mmol) at 0° C. under N₂. After 14 hours, the reaction mixture wasdiluted with H₂O (50 mL) and extracted with EtOAc (2×100 mL). Theorganic layers were combined, dried over anhydrous MgSO₄, filtered andconcentrated. The crude product was purified by column chromatography toproduce the compound 14b (4.3 g, 69%). ¹H-NMR (400 MHz, CDCl₃) δ 3.59(t, J=6.4 Hz, 2H), 3.17 (br s, 2H), 2.82 (s, 3H), 1.62-1.21 (m, 17H),0.88 (s, 9H), 0.04 (s, 6H).

Preparation of Compound 14c

To a solution of compound 14b (4.3 g, 12.4 mmol) in THF (15 mL) wasadded TBAF (1 M in THF, 15 mL, 14.9 mmol) at 0° C. under N₂. After 5hours, the reaction mixture was diluted with H₂O (50 mL) and extractedwith diethyl ether (2×100 mL). The organic layers were combined, driedover anhydrous MgSO₄, filtered and concentrated. The crude product waspurified by column chromatography to produce the compound 14c (3.0 g,98%). ¹H-NMR (400 MHz, CDCl₃) δ 3.63 (br s, 2H), 3.20 (br s, 2H), 2.82(s, 3H), 1.65-1.23 (m, 17H).

Preparation of Compound 14d

To a solution of compound 14c (3.0 g, 12.9 mmol) in THF (30 mL) wasadded carbon tetrabromide (6.4 g, 19.4 mmol) and triphenylphosphine (5.1g, 19.4 mmol) at 0° C. under N₂. After 2 hours, the reaction mixture wasfiltered through silica gel and washed diethyl ether (100 mL). Thefiltrate was concentrated and purified by column chromatography toproduce the compound 14d (3.3 g, 86%). ¹H-NMR (400 MHz, CDCl₃) δ 3.40(t, J=6.8 Hz, 2H), 3.19 (br s, 2H), 2.83 (s, 3H), 1.90-1.70 (m, 2H),1.65-1.40 (m, 13H), 1.38-1.25 (m, 2H).

Preparation of Compound 14e

DIPEA (53.0 mL, 302.5 mmol) and EDC.HCl (35.7 g, 186.2 mmol) were addedto a stirred mixture of compound 2d (35.0 g, 116.4 mmol) and5-formylsalicylic acid (21.3 g, 128.0 mmol) in DCM (1.6 L) at 0° C. Thereaction mixture was stirred at room temperature for 20 hours under N₂.The reaction mixture was diluted with saturated aq. NH₄Cl solution (1.5L) and extracted DCM (2×1.5 L). The combined organic layers washed withbrine (1.5 L) and dried anhydrous MgSO₄, filtered and concentrated. Thecrude product was purified by column chromatography to produce thecompound 14e (28.2 g, 59%). ¹H-NMR (400 MHz, CDCl₃) δ 13.37 (br s, 1H),9.86 (s, 1H), 8.20 (s, 1H), 8.07 (br s, 2H), 7.90 (d, J=8.4 Hz, 1H),7.07 (d, J=8.4 Hz, 1H), 4.06-4.01 (m, 2H), 3.79-3.66 (m, 10H), 1.47 (s,9H).

Preparation of Compound 14f

To a solution of compound 14e (28.0 g, 67.9 mmol) in MeCN (500 mL) wereadded compound M (29.7 g, 74.7 mmol), 4 Å molecular sieve (30 g) andAg₂O (62.9 g, 272 mmol). After stirring at room temperature for 12 hoursunder N₂, the reaction mixture was concentrated, diluted with H₂O (800mL) and extracted with EtOAc (1 L). The combined organic layers weredried over anhydrous MgSO₄, filtered and concentrated. The residue waspurified by column chromatography to produce the compound 14f (30.1 g,61%). ¹H-NMR (400 MHz, CDCl₃) δ 9.99 (s, 1H), 8.54 (s, 1H), 7.99 (d,J=8.8 Hz, 1H), 7.68 (s, 1H), 7.44 (br s, 1H), 7.18 (d, J=8.8 Hz, 1H),5.45-5.30 (m, 4H), 4.26 (d, J=9.2 Hz, 1H), 4.02-3.97 (m, 2H), 3.80-3.55(m, 13H), 2.06 (s, 9H), 1.46 (s, 9H).

Preparation of Compound 14g

To a solution of compound 14f (29.0 g, 39.8 mmol) in i-PrOH/CHCl₃ (90mL/450 mL) was added silica gel (16.7 g) and NaBH₄ (3.70 g, 99.5 mmol)at 0° C. After stirring at 0° C. for 2 hours under N₂, the reactionmixture was quenched with H₂O (500 mL) and extracted with EtOAc (1 L).The organic layer was dried over anhydrous MgSO₄, filtered andconcentrated. The crude product was purified by column chromatography toproduce the compound 14g (24.1 g, 83%). ¹H-NMR (400 MHz, CDCl₃) δ 7.98(s, 1H), 7.72 (s, 1H), 7.46 (d, J=8.8 Hz, 1H), 7.41 (br, 1H), 7.04 (d,J=8.8 Hz, 1H), 5.41-5.24 (m, 4H), 4.67 (d, J=6.6 Hz, 2H), 4.19 (d, J=8.8Hz, 1H), 3.99-3.93 (m, 2H), 3.79-3.65 (m, 12H), 3.59-3.50 (m, 1H),2.08-2.00 (m, 10H), 1.46 (s, 9H).

Preparation of Compound 14h

To a solution of compound 14g (23.7 g, 31.5 mmol) in DMF (50 mL) wereadded bis(4-nitrophenyl)carbonate (8.9 g, 29.3 mmol) and DIPEA (5.65 mL,31.5 mmol) at 0° C. under N₂. The reaction mixture was stirred at 0° C.for 30 minutes and allowed to warm to room temperature for 1 hour. Thereaction mixture was diluted with H₂O (500 mL) and extracted with EtOAc(500 mL). The organic layer was washed with brine (2×200 mL), dried overanhydrous MgSO₄, filtered, and concentrated. The crude product waspurified by column chromatography to produce the compound 14h (22.4 g,77%) as white foam. ¹H-NMR (400 MHz, CDCl₃) δ 8.28 (d, J=7.2 Hz, 2H),8.13 (s, 1H), 7.68 (br s, 1H), 7.52 (d, J=8.8 Hz, 1H), 7.47 (br, 1H),7.38 (d, J=7.2 Hz, 2H), 7.08 (d, J=8.8 Hz, 1H), 5.44-5.24 (m, 6H), 4.21(d, J=9.6 Hz, 1H), 4.00 (br s, 2H), 3.80-3.64 (m, 12H), 3.64-3.54 (m,1H), 2.06 (s, 9H), 1.47 (s, 9H).

Preparation of Compound 14i

α-Amanitin (60.0 mg, 0.065 mmol) was dissolved in DMSO (2 mL) andcompound 14d (114 mg, 0.39 mmol) and potassium tert-butoxide (0.065 mL,0.065 mmol) were added at 0° C. under N₂. After 4 hours at 0° C., the pHof the solution was adjusted to 4-5 with acetic acid. The residue wasdissolved in DMSO (1 mL) and purified by HPLC, which produced thecompound 14i (29 mg, 39%) as white solid. EI-MS m/z: [M-Boc]⁺ 1032.4.

Preparation of Compound 14j

To a solution of compound 14i (29 mg, 0.026 mmol) in DCM (3 mL) wasadded TFA (0.5 mL) at 0° C. After 2 hours at 0° C., the solvent andexcess TFA were removed by N₂ flow and the resulting residue waspurified by HPLC, which produced the compound 14j (26 mg, 99%) as whitesolid. EI-MS m/z: [M+H]⁺ 1032.3, [M+Na]⁺ 1054.3.

Preparation of Compound 14k

Compound 14j (13 mg, 0.011 mmol), compound 14h (10 mg, 0.011 mmol) andanhydrous HOBt (0.3 mg, 0.002 mmol) were dissolved in DMF (0.5 mL) at 0°C. Then pyridine (0.2 mL) and DIPEA (0.004 mL, 0.023 mmol) were added.After stirring at room temperature for 24 hours under N₂, the reactionmixture was dissolved in DMSO (1 mL) and purified by HPLC, whichproduced the compound 14k (11 mg, 54%). EI-MS m/z: [M+H]⁺ 1788.1.

Preparation of Compound 14l

To a solution of compound 14k (11 mg, 0.006 mmol) in MeOH (0.2 mL) wasadded LiOH monohydrate (1.3 mg, 0.03 mmol) in H₂O (0.2 mL) at −20° C.After 1 hour at 0° C., the pH of the solution was adjusted to 4-5 withacetic acid. The resulting solution was dissolved in DMSO (1 mL) andpurified by HPLC, which produced the compound 14l (7.5 mg, 75%) as whitesolid. EI-MS m/z: [M+H]⁺ 1648.6.

Preparation of Compound 14m

To a solution of compound 14l (7.5 mg, 0.0045 mmol) in DCM (3 mL) wasadded TFA (0.5 mL) at 0° C. After 2 hours at 0° C., the solvent andexcess TFA were removed by N₂ flow. Then the residue was purified byHPLC, which produced the compound 14m (6.2 mg, 85%) as white solid.EI-MS m/z: [M+H]⁺: 1548.5.

Example 24. Preparation of Compound 15b

Preparation of Compound 15a

Compound 15a was prepared from compound 3e by a method similar to methodof preparing compound 14h of Example 23. EI-MS m/z: [M+H]⁺ 1128.3,[M+H-Boc]⁺ 1028.3, [M+H−2Boc]⁺ 928.2.

Preparation of Compound 15b

Compound 15b was prepared from compound 14j and compound 15a by a methodsimilar to method of preparing compound 14m of Example 23. EI-MS m/z:[M+H]⁺ 1681.6.

Example 25. Preparation of Compound 16f

Preparation of Compound 16a

To a stirred solution of oxalyl chloride (2.8 mL, 32.5 mmol) in DCM (5mL) DMSO (3.08 mL, 43.4 mmol) was added in DCM (15 mL) and then thereaction mixture was stirred at −78° C. for 30 minutes. To this solutionwas added compound 2a (3.8 g, 21.7 mmol) at −78° C. and stirred for 1hour. Triethylamine (15.1 mL, 108 mmol) in DCM (20 mL) was added andthen the reaction mixture was allowed to warm to room temperature,diluted with H₂O (100 mL) and extracted with DCM (2×100 mL). The organiclayers were combined, dried over anhydrous MgSO₄, filtered andconcentrated. The residue was purified by column chromatography toproduce the compound 16a (1.8 g, 48%). ¹H-NMR (400 MHz, CDCl₃) δ 9.74(s, 1H), 4.19 (s, 2H), 3.77-3.69 (m, 6H), 3.42 (m, 2H).

Preparation of Compound 16b

To a solution of compound 16a (1.0 g, 3.32 mmol) and compound 2d (1.72g, 9.96 mmol) in MeOH (15 mL) AcOH (0.19 mL, 3.32 mmol) was added at 0°C. After stirring for 30 minutes at 0° C., NaCNBH₃ (658 mg, 9.96 mmol)was added and allowed to warm to room temperature over 2 hours. Afterthe reaction was completed, the reaction mixture was diluted with H₂O(50 mL) and then extracted with DCM (3×100 mL). The organic layers werecombined, dried over anhydrous MgSO₄, filtered and concentrated. Theresidue was purified by column chromatography to produce the compound16b (800 mg, 41%) as light yellowish oil. ¹H-NMR (400 MHz, CDCl₃) δ 7.78(brs, 1H), 4.01 (m, 2H), 3.69-3.65 (m, 24H), 3.39 (m, 4H), 3.04 (m, 6H),1.47 (s, 9H).

Preparation of Compound 16c

To a solution of compound 16b (350 mg, 0.60 mmol) in MeOH (10 mL) Pd/C(10 wt. %, 300 mg) was added. After stirring at room temperature for 8hours under hydrogen, the reaction mixture was filtered through a celitepad and washed with MeOH (100 mL). Concentration provided compound 16cas colorless oil (300 mg, 94%), which was used without furtherpurification. ¹H-NMR (400 MHz, CDCl₃) δ 4.02 (m, 2H), 3.71 (m, 2H),3.65-3.55 (m, 22H), 2.92 (m, 4H), 2.76 (t, J=5.2 Hz, 6H), 1.47 (s, 9H).EI-MS m/z: [M+H]⁺ 527.6.

Preparation of Compound 16d

DIPEA (0.40 mL, 2.24 mmol) and PyBOP (711 mg, 1.34 mmol) were added to astirred mixture of compound 1j (1.57 g, 1.23 mmol) and compound 16c (300mg, 0.56 mmol) in DMF (15 mL). After stirring at room temperature for 4hours under N₂, the reaction mixture was diluted H₂O (200 mL) andextracted with EtOAc (3×100 mL). The combined organic layers were driedover anhydrous Na₂SO₄, filtered and concentrated under reduced pressure.The residue was dissolved in H₂O/DMSO (5 mL/5 mL) and purified by HPLCproduced the compound 16d (1.57 g, 91.8%). EI-MS m/z: 1/2[M+H]⁺ 1502.7.

Preparation of Compound 16f

To a solution of compound 16d (1.10 g, 0.36 mmol) in MeOH/THF (5 mL/10mL) NaOH (175 mg, 4.32 mmol) was added dropwise in H₂O (3 mL) at 0° C.After 3 hours at 0° C., the pH of the solution was adjusted to pH 4using 2 N aq. HCl and concentrated. The residue was diluted with DCM (12mL) and TFA (3 mL) at 0° C. After 2 hours at 0° C., the solvent andexcess TFA were removed by N₂ flow. The residue was dissolved inH₂O/MeCN (7.5 mL/7.5 mL) and purified by HPLC produced the compound 16f(432 mg, 46%) as white solid. EI-MS m/z: 1/2[M+H]⁺ 1298.5.

Example 26. Preparation of Compound 16g

Compound 16g was prepared from compound 1i and compound 16c by a similarmethod of preparing compound 16f in Example 25. EI-MS m/z: 1/2[M+H]⁺1284.5.

Example 27. Preparation of Compound 17d

Preparation of Compound 17a

To a stirred solution of oxalyl chloride (0.62 mL, 7.3 mmol) in DCM (4mL) DMSO (1.04 mL, 14.6 mmol) was added in DCM (10 mL) and then thereaction mixture was stirred at −78° C. for 30 minutes. To this solutionwas added compound 3b (1.5 g, 4.88 mmol) at −78° C. and stirred for 1hour. Triethylamine (2.72 mL, 19.50 mmol) in DCM (7 mL) was added andthen the reaction mixture was allowed to warm to room temperature. Afterconcentration under reduced pressure, the residue was purified by columnchromatography (EtOAc to EtOAc/MeOH 10/1), which produced the compound17a (1.23 g, 82%). ¹H-NMR (400 MHz, CDCl₃) δ 9.73 (s, 1H), 4.16 (s, 2H),3.75-3.61 (m, 18H), 3.39 (t, J=5.2 Hz, 2H).

Preparation of Compound 17b

NaCNBH₃ (257 mg, 4.09 mmol) was added to a stirred mixture of compound17a (1.30 g, 4.25 mmol) and compound 2d (492 mg, 1.63 mmol) in MeOH (5mL) at 0° C. The reaction mixture was then gradually heated up to roomtemperature over 2 hours. After the reaction was completed, the reactionmixture was concentrated under reduced pressure. The residue waspurified by column chromatography (EtOAc to EtOAc/MeOH 10/1), whichproduced the compound 17b (620 mg, 45%).

¹H-NMR (400 MHz, DMSO-d₆) δ 9.96 (br, 1H), 3.79 (t, J=4.8 Hz, 2H) 3.59(t, J=4.8 Hz, 4H), 3.56-3.46 (m, 38H), 3.44-3.37 (m, 10H), 2.66-2.56 (m,6H), 1.39 (s, 9H).

Preparation of Compound 17c

To a solution of compound 17b (300 mg, 0.35 mmol) in MeOH (7 mL) wasadded Pd/C (10 wt. %, 38 mg). After stirring at room temperature for 4hours under hydrogen, the reaction mixture was filtered through a celitepad and washed with MeOH (400 mL). Concentration provided compound 17cas colorless oil (253 mg, 90%), which was used without furtherpurification. ¹H-NMR (400 MHz, DMSO-d₆) δ 3.79 (t, J=4.4 Hz, 2H),3.55-3.45 (m, 38H), 3.42 (t, J=6.0 Hz, 10H), 2.66-2.56 (m, 10H), 1.39(s, 9H). EI-MS m/z: [M+H]⁺ 791.0.

Preparation of Compound 17d

Compound 17d was prepared from compound 1i and compound 17c by a similarmethod of preparing compound 16f in Example 25. EI-MS m/z: 1/2[M+H]⁺1415.6.

Example 28. Preparation of Compound 18c

Preparation of Compound 18a

NaCNBH₃ (197 mg, 3.14 mmol) was added to a stirred mixture of compound17a (998 mg, 3.26 mmol) and compound 3e (670 mg, 1.25 mmol) in MeOH (4mL) at 0° C. The reaction mixture was then gradually heated up to roomtemperature over 2 hours. After the reaction was completed, the reactionmixture was concentrated under reduced pressure. The residue waspurified by column chromatography (EtOAc to EtOAc/MeOH 10/1), whichproduced the compound 18a (668 mg, 49%). ¹H-NMR (400 MHz, DMSO-d₆) δ3.97 (m, 2H) 3.63-3.57 (m, 6H), 3.56-3.44 (m, 46H), 3.44-3.36 (m, 12H),2.66-2.61 (m, 6H), 1.45 (s, 18H).

Preparation of Compound 18b

To a solution of compound 18a (60 mg, 0.055 mmol) in MeOH (1.2 mL) Pd/C(10 wt. %, 6 mg) was added. After stirring at room temperature for 4hours under hydrogen, the reaction mixture was filtered through a celitepad and washed with MeOH (400 mL). Concentration provided compound 18b(55 mg, 96%) as colorless oil, which was used without furtherpurification. ¹H-NMR (400 MHz, DMSO-d₆) δ 3.97 (m, 2H), 3.62-3.57 (m,4H), 3.54-3.45 (m, 50H), 3.45-3.39 (m, 10H), 2.66-2.61 (m, 10H), 1.46(s, 18H). EI-MS m/z: 1/2[M+H]⁺ 1023.3.

Compound 18c was prepared from compound 1i and compound 18b by a similarmethod of preparing compound 16f in Example 25. EI-MS m/z: 1/2[M+H]⁺1481.7.

Example 29. Preparation of Compound 19c

Preparation of Compound 19a

NaCNBH₃ (197 mg, 3.14 mmol) was added to a stirred mixture of compound17a (118 mg, 0.16 mmol) and compound 4e (232 mg, 0.76 mmol) in MeOH (1mL) at 0° C. The reaction mixture was then gradually heated up to roomtemperature over 2 hours. After the reaction was completed, the reactionmixture was evaporated under reduced pressure. The residue was purifiedby column chromatography (EtOAc to EtOAc/MeOH 10/1), which produced thecompound 19a (135 mg, 68%). ¹H-NMR (400 MHz, DMSO-d₆) δ 7.72 (br s, 1H)4.02 (t, 2H), 3.72-3.53 (m, 86H), 3.39 (t, 4H), 2.77 (bs, 4H), 1.47 (s,9H). EI-MS m/z: [M+H]⁺ 1239.6.

Preparation of Compound 19b

To a solution of compound 19a (133 mg, 0.107 mmol) in MeOH (2 mL) Pd/C(10 wt. %, 26 mg) was added and HCl (4 N in 1,4-dioxane, 0.054 mL, 0.21mmol) at 0° C. After stirring at room temperature for 40 minutes underhydrogen, the reaction mixture was filtered through a celite pad andwashed with MeOH (40 mL). Concentration provided compound 19b (132 mg,97%) as colorless oil, which was used without further purification.¹H-NMR (400 MHz, DMSO-d₆) δ 7.79 (s, 1H), 4.06-4.02 (m, 8H), 3.88 (m,2H), 3.73-3.64 (m, 80H), 3.22 (s, 4H), 1.47 (s, 9H). EI-MS m/z: [M+H]⁺:1187.5.

Preparation of Compound 19c

Compound 19c was prepared from compound 1i and compound 19b by a similarmethod of preparing compound 16f in Example 25. EI-MS m/z: 1/2[M+H]⁺1614.5.

Example 30. Preparation of Compound 20q

Preparation of Compound 20a

To a solution of 2-(2-(2-chloroethoxy)ethoxy)ethanol (5.0 g, 29.6 mmol)in acetone (30 mL) was added NaI (13.3 g, 88.9 mmol). The reactionmixture was refluxed for 12 hours. After the reaction was completed, thereaction mixture was filtered and concentrated. The crude product waspurified by column chromatography to produce the compound 20a (7.0 g,91%). ¹H-NMR (400 MHz, CDCl₃) δ 3.80-3.73 (m, 4H), 3.72-3.65 (m, 4H),3.63-3.61 (m, 2H), 3.27 (t, J=6.4 Hz, 2H).

Preparation of Compound 20b

To a solution of compound 20a (7.0 g, 26.9 mmol) in acetone (200 mL) at0° C. Jones reagent (20 mL) was added. After 15 hours at 0° C., thereaction mixture was filtered and concentrated. The residue was dilutedwith H₂O (150 mL) and extracted with EtOAc (2×100 mL). The organiclayers were combined, dried over anhydrous MgSO₄, filtered andconcentrated. The crude product was purified by column chromatography toproduce the compound 20b (7.0 g, 94%). ¹H-NMR (400 MHz, CDCl₃) δ 4.22(s, 2H), 3.85-3.70 (m, 6H), 3.35-3.25 (m, 2H).

Preparation of Compound 20c

To a solution of compound 20b (7.0 g, 25.5 mmol) in MeOH (70 mL) oxalylchloride (3.2 mL, 38.3 mmol) was added at 0° C. under N₂. After 16hours, the reaction mixture was concentrated and purified by columnchromatography, which produced the compound 20c (5.7 g, 77%). ¹H-NMR(400 MHz, CDCl₃) δ 4.19 (s, 2H), 3.80-3.67 (m, 9H), 3.27 (t, J=6.8 Hz,2H).

Preparation of Compound 20d

To a solution of compound 20c (2.5 g, 8.67 mmol) andN,N-diBoc-hydroxylamine (2.6 g, 11.2 mmol) in DMF (30 mL) was added NaH(60% in oil, 454 mg, 10.4 mmol) at 0° C. under N₂. After 15 hours, thereaction mixture was diluted with H₂O (50 mL) and extracted with EtOAc(3×100 mL). The organic layers were combined, dried over anhydrousMgSO₄, filtered and concentrated. The crude product was purified bycolumn chromatography to produce the compound 20d (1.87 g, 73%). ¹H-NMR(400 MHz, CDCl₃) δ 4.17 (s, 2H), 4.08 (m, 2H), 3.78-3.65 (m, 9H), 1.53(s, 18H).

Preparation of Compound 20e

To a solution of compound 20d (1.87 g, 6.38 mmol) in THF/MeOH/H₂O (45mL/15 mL/15 mL) NaOH (600 mg, 15.9 mmol) was added at 0° C. under N₂.The reaction mixture was stirred for 3 hours at room temperature. Thenthe pH of the solution was adjusted to 4-5 with 1 N aqueous HCl. Thereaction mixture was poured into H₂O (100 mL) and extracted with EtOAc(2×100 mL). The organic layers were combined, dried over MgSO₄, filteredand concentrated. The compound 20e (1.6 g, 90%) was produced ascolorless oil, and it was used without further purification. ¹H-NMR (400MHz, CDCl₃) δ 4.17 (s, 2H), 4.08-4.02 (m, 2H), 3.80-3.67 (m, 6H), 1.48(s, 9H).

Preparation of Compound 20f

Pd/C (10 wt. %, 1.0 g) was added to a solution of compound 2a (6.7 g,38.2 mmol) in MeOH (38 mL). After stirring at room temperature for 8hours under hydrogen, the reaction mixture was filtered through a celitepad and washed with MeOH (100 mL). Concentration provided compound 20f(5.6 g, 99%) as colorless oil, which was used without furtherpurification. ¹H-NMR (400 MHz, CDCl₃) δ 3.95-3.25 (m, 12H), 2.90 (s,2H).

Preparation of Compound 20g

Benzyl chloroformate (6 mL, 42.2 mmol) were slowly added to a solutionof compound 20f (5.6 g, 38.2 mmol) and triethylamine (8 mL, 57.6 mmol)in THF (200 mL) at 0° C. for 30 minutes under N₂. After stirring for 1hour at 0° C., the reaction mixture was concentrated and the crudeproduct was purified by column chromatography, which produced thecompound 20g (5.7 g, 53%). ¹H-NMR (400 MHz, CDCl₃) δ 7.45-7.20 (m, 5H),5.61 (br s, 1H), 5.07 (s, 2H), 3.85-3.20 (m, 12H).

Preparation of Compound 20h

To a solution of compound 20g (2.7 g, 9.53 mM) in DCM (30 mL) were addedtriethylamine (1.9 mL, 12.3 mmol) and p-toluenesulfonyl chloride (2.3 g,10.4 mmol) at room temperature under N₂. After 8 hours, the reactionmixture was diluted with H₂O (50 mL) and extracted with DCM (3×100 mL).The organic layers were combined, dried over anhydrous MgSO₄, filteredand concentrated. The crude product was purified by columnchromatography to produce the compound 20h (3.51 g, 84%). ¹H-NMR (400MHz, CDCl₃) δ 7.78 (d, J=7.2 Hz, 2H), 7.45-7.25 (m, 7H), 5.20 (br s,1H), 5.09 (s, 2H), 4.20-4.05 (m, 2H), 3.75-3.25 (m, 10H), 2.43 (s, 3H).

Preparation of Compound 20i

A solution of compound 20h (3.51 g, 8.02 mmol) and NaN₃ (3.8 g, 57.6mmol) in DMF (27 mL) was heated at 100° C. for 15 hours. After thereaction was completed, the reaction mixture was filtered andconcentrated. The residue was diluted with H₂O (50 mL) and extractedwith EtOAc (2×100 mL). The organic layers were combined, dried overanhydrous MgSO₄, filtered and concentrated. The crude product waspurified by column chromatography to produce the compound 20i (2.05 g,83%). ¹H-NMR (400 MHz, CDCl₃) δ 7.45-7.25 (m, 5H), 5.20 (br s, 1H), 5.10(s, 2H), 3.80-3.25 (m, 12H).

Preparation of Compound 20j

Triphenylphosphine (2.09 g, 7.97 mmol) was added to a solution ofcompound 20i (2.05 g, 6.64 mmol) in THF (27 mL) at room temperature.After stirring for 2 hours under N₂, H₂O (0.6 mL, 33.2 mmol) was addedand the reaction mixture was refluxed for 3 hours. Then the reactionmixture was concentrated and purified by column chromatography, whichproduced the compound 20j (1.78 g, 95%). ¹H-NMR (400 MHz, CDCl₃) δ7.45-7.25 (m, 5H), 5.63 (br s, 1H), 5.10 (s, 2H), 3.80-3.25 (m, 10H),2.88 (s, 2H).

Preparation of Compound 20k

To a stirred solution of oxalyl chloride (1.4 mL, 15.9 mmol) in DCM (14mL) was added DMSO (2.3 mL, 31.9 mmol) in DCM (28 mL) and then thereaction mixture was stirred at −78° C. for 30 minutes. To this solutionwas added compound 20g (3.01 g, 10.6 mmol) at −78° C. After stirring for1 hour at −78 at 0° C., triethylamine (7.4 mL, 53.1 mmol) was added andthe reaction was allowed to warm to room temperature. The reactionmixture was poured into H₂O (100 mL) and extracted with EtOAc (2×100mL). The organic layers were combined, dried over MgSO₄. Filtration andconcentration produced the compound 20k (2.6 g), which was used withoutfurther purification. ¹H-NMR (400 MHz, CDCl₃) δ 9.70 (s, 1H), 7.45-7.25(m, 5H), 5.25 (br s, 1H), 5.10 (s, 2H), 3.80-3.25 (m, 10H).

Preparation of Compound 20l

To a solution of compound 20j (1.78 g, 6.30 mmol) and compound 20k (2.13g, 7.56 mmol) in MeOH (63 mL) was added NaCNBH₃ (674 mg, 10.7 mmol) atroom temperature under N₂. After 3 hours, the reaction mixture wasfiltered and concentrated. The crude product was purified by columnchromatography to produce the compound 20l (2.01 g, 58%). ¹H-NMR (400MHz, CDCl₃) δ 7.45-7.25 (m, 10H), 5.60 (br s, 2H), 5.03 (s, 4H),3.80-3.25 (m, 20H), 2.81 (s, 4H).

Preparation of Compound 20m

DIPEA (0.4 mL, 2.28 mmol) and PyBOP (713 mg, 1.36 mmol) were added to astirred solution of compound 20l (500 mg, 0.91 mmol) and compound 20e(306 mg, 1.09 mmol) in DMF (10 mL). After stirring at room temperaturefor 6 hours under N₂, the reaction mixture was diluted water (100 mL)and extracted with EtOAc (3×100 mL). The combined organic layers weredried over anhydrous Na₂SO₄, filtered and concentrated under reducedpressure. The crude product was purified by column chromatography toproduce the compound 20m (318 mg, 43%). ¹H-NMR (400 MHz, CDCl₃) δ7.45-7.25 (m, 10H), 5.47 (br s, 1H), 5.37 (br s, 1H), 5.09 (s, 4H),3.80-3.25 (m, 34H), 1.46 (s, 9H). EI-MS m/z: [M+H]⁺ 808.9.

Preparation of Compound 20n

To a solution of compound 20m (318 mg, 0.39 mmol) in MeOH (30 mL) wasadded Pd/C (10 wt. %, 1.0 g). After stirring at room temperature for 3hours under hydrogen, the reaction mixture was filtered through a celitepad and washed with MeOH (100 mL). Concentration provided compound 20n(180 mg) as colorless oil, which was used without further purification.EI-MS m/z: [M+H]⁺ 541.2.

Preparation of Compound 20o

DIPEA (0.034 mL, 0.19 mmol) and PyBOP (63 mg, 0.12 mmol) were added to astirred solution of compound 1i (130 mg, 0.10 mmol) and compound 20n (26mg, 0.04 mmol) in DMF (3 mL) at 0° C. After stirring at 0° C. for 30minutes, the reaction was allowed to warm to room temperature over 20hours under N₂. The reaction mixture was poured into H₂O (50 mL) andextracted with EtOAc (3×50 mL). The organic layers were combined,filtered and concentrated under reduced pressure. The residue wasdissolved in DMSO (1 mL) and purified by HPLC, which produced thecompound 20o (28 mg, 10%) as white solid. EI-MS m/z: 1/2[M+H]⁺ 1481.5,1/2[M+Na]⁺ 1503.8.

Preparation of Compound 20p

To a solution of compound 20o (28 mg, 0.009 mmol) in MeOH (1 mL) wasadded LiOH monohydrate (2 mg, 0.047 mmol) in H₂O (1 mL) at −5° C. Thereaction mixture was stirred at −5° C. for 1 hour. After the reactionwas completed, the pH of the solution was adjusted to 4-5 with aceticacid. The residue was dissolved in DMSO (1 mL) and purified by HPLC,which produced the compound 20p (16 mg, 67%) as white solid. EI-MS m/z:1/2[M+H]⁺: 1341.4.

Preparation of Compound 20q

To a solution of compound 20p (16 mg, 0.0059 mmol) in DCM (2 mL) wasadded TFA (0.2 mL) at 0° C. After 2 hours at 0° C., the solvent andexcess TFA were removed by N₂ flow. The residue was dissolved in DMSO (1mL) and purified by HPLC, which produced the compound 20q (8.5 mg, 56%)as white solid. EI-MS m/z: 1/2[M+H]⁺: 1291.3.

Example 31. Preparation of Compound 21i

Preparation of Compound 21a

To a solution of compound 3b (9.0 g, 29.2 mmol) in MeOH (146 mL) wasadded Pd/C (10 wt. %, 3.0 g) and the reaction mixture was stirred atroom temperature for 5 hours under hydrogen. Then the reaction mixturewas filtered through a celite pad and washed with MeOH (100 mL).Concentration provided compound 21a (8.2 g, 100%) as colorless oil,which was used without further purification. ¹H-NMR (400 MHz, CDCl₃) δ3.80-3.60 (m, 24H), 3.01 (t, J=4.8 Hz, 2H).

Preparation of Compound 21b

To a solution of compound 21a (8.24 g, 29.2 mmol) in THF (190 mL) wasadded triethylamine (6.1 mL, 43.9 mmol) and benzyl chloroformate (4.6 mL32.2 mmol) at 0° C. under N₂. The reaction mixture was concentrated andthe crude product was purified by column chromatography to produce thecompound 21b (5.59 g, 46%). ¹H-NMR (400 MHz, CDCl₃) δ 7.45-7.20 (m, 5H),5.61 (br s, 1H), 5.09 (s, 2H), 3.85-3.50 (m, 22H), 3.39 (m, 2H).

Preparation of Compound 21c

To a solution of compound 21b (3.09 g, 7.43 mmol) in THF (75 mL) wereadded 4-methylmorpholine (1.1 mL, 9.66 mmol) and methanesulfonicanhydride (1.43 g, 8.18 mmol) at 0° C. under N₂. After 5 hours at 0° C.,NaN₃ (969 mg, 14.9 mmol) and DMF (20 mL) were added. After 16 hoursunder reflux, the reaction mixture was filtered and concentrated. Theresidue was diluted with H₂O (50 mL) and extracted with EtOAc (2×100mL). The organic layers were combined, dried over anhydrous MgSO₄,filtered and concentrated. The crude product was purified by columnchromatography to produce the compound 21c (2.62 g, 80%). ¹H-NMR (400MHz, CDCl₃) δ 7.45-7.20 (m, 5H), 5.45 (br s, 1H), 5.09 (s, 2H),3.85-3.25 (m, 24H).

Preparation of Compound 21d

Triphenylphosphine (1.87 g, 7.13 mmol) was added to a solution ofcompound 21c (2.62 g, 5.94 mmol) in THF (30 mL) at room temperature.After stirring for 2 hours under N₂, H₂O (0.54 mL, 29.7 mmol) was addedand the reaction mixture was refluxed for 3 hours. The reaction mixturewas concentrated and purified by column chromatography, which producedthe compound 21d (2.47 g, 95%). ¹H-NMR (400 MHz, CDCl₃) δ 7.45-7.25 (m,5H), 5.63 (br s, 1H), 5.09 (s, 2H), 3.80-3.25 (m, 22H), 3.00-2.80 (m,2H).

Preparation of Compound 21e

To a stirred solution of oxalyl chloride (0.78 mL, 9.02 mmol) in DCM (14mL) was added DMSO (1.3 mL, 18.1 mmol) in DCM (6 mL) and then thereaction mixture was stirred at −78° C. for 30 minutes. To this solutionwas added compound 21b (2.5 g, 6.01 mmol) at −78° C. After stirred for 1hour at −78° C., triethylamine (4.2 mL, 30.1 mmol) was added and thereaction was allowed to warm to room temperature. The reaction mixturewas poured into H₂O (100 mL) and extracted with EtOAc (2×100 mL). Theorganic layers were combined, dried over MgSO₄. Filtration andconcentration produced the compound 21e (2.29 g), which was used withoutfurther purification. ¹H-NMR (400 MHz, CDCl₃) δ 9.70 (s, 1H), 7.45-7.25(m, 5H), 5.25 (br s, 1H), 5.10 (s, 2H), 3.80-3.25 (m, 24H).

Preparation of Compound 21f

To a solution of compound 21d (2.47 g, 5.95 mmol) and compound 21e (2.29g, 5.52 mmol) in MeOH (50 mL) was added NaCNBH₃ (530 mg, 8.44 mmol) atroom temperature under N₂. After 3 hours, the reaction mixture wasfiltered and concentrated. The crude product was purified by columnchromatography to produce the compound 21f (2.05 g, 51%). ¹H-NMR (400MHz, CDCl₃) δ 7.45-7.25 (m, 10H), 5.47 (br s, 1H), 5.37 (br s, 1H), 5.09(s, 4H), 3.80-3.25 (m, 48H).

Preparation of Compound 21g

DIPEA (0.27 mL, 1.53 mmol) and HBTU (350 mg, 0.92 mmol) were added to astirred solution of compound 21f (380 mg, 0.61 mmol) and compound 20e(206 mg, 0.73 mmol) in DMF (6 mL). After stirring at room temperaturefor 6 hours under N₂, the reaction mixture was diluted water (100 mL)and extracted with EtOAc (3×100 mL). The combined organic layers weredried over anhydrous Na₂SO₄, filtered and concentrated under reducedpressure. The crude product was purified by column chromatography toproduce the compound 21g (210 mg, 42%). ¹H-NMR (400 MHz, CDCl₃) δ7.45-7.25 (m, 10H), 5.47 (br s, 1H), 5.37 (br s, 1H), 5.09 (s, 4H),3.80-3.25 (m, 34H), 1.46 (s, 9H).

Preparation of Compound 21h

To a solution of compound 21g (210 mg, 0.19 mmol) in MeOH (30 mL) wasadded Pd/C (10 wt. %, 1.0 g) and then the reaction mixture was stirredat room temperature for 4 hours under hydrogen. The reaction mixture wasfiltered through a celite pad and washed with MeOH (50 mL).Concentration provided compound 21h (30 mg) as colorless oil, which wasused without further purification. EI-MS m/z: [M+H]⁺ 805.2, [M+Na]⁺827.2.

Preparation of Compound 21i

Compound 21i was prepared from compound 1i and compound 21h by a similarmethod of preparing compound 20q in Example 30. EI-MS m/z: 1/2[M+H]⁺1423.7, 1/2[M+Na]⁺ 1445.2.

Example 32. Preparation of Compound 22h

Preparation of Compound 22a

To a solution of compound 3a (8.0 g, 18.3 mmol) in THF (50 mL) was addedLiBr (7.9 g, 91.6 mmol) at room temperature. After stirring for 17 hoursunder reflux, the reaction mixture was filtered and concentrated. Thecrude product was purified by column chromatography to produce thecompound 22a (3.2 g, 50%). ¹H-NMR (400 MHz, CDCl₃) δ 3.95-3.50 (m, 24H).

Preparation of Compound 22b

To a solution of compound 22a (3.2 g, 12.3 mmol) in acetone (20 mL) at0° C. was added Jones reagent (20 mL). After 15 hours at 0° C., thereaction mixture was filtered and concentrated. The residue was dilutedwith H₂O (50 mL) and extracted with EtOAc (2×100 mL). The organic layerswere combined, dried over anhydrous MgSO₄, filtered and concentrated.The crude product was purified by column chromatography to produce thecompound 22b (3.2 g, 72%). ¹H-NMR (400 MHz, CDCl₃) δ 4.16 (s, 2H),3.95-3.30 (m, 20H).

Preparation of Compound 22c

To a solution of compound 22b (3.2 g, 8.90 mmol) in MeOH (30 mL) wasadded oxalyl chloride (1.15 mL, 13.3 mmol) at 0° C. under N₂. After 16hours, the reaction mixture was concentrated and purified by columnchromatography, which produced the compound 22c (2.7 g, 81%). ¹H-NMR(400 MHz, CDCl₃) δ 4.17 (s, 2H), 3.80-3.60 (m, 21H), 3.47 (t, J=6.4 Hz,2H).

Preparation of Compound 22d

NaH (60% in oil, 378 mg, 8.63 mmol) was added to a solution of compound22c (2.7 g, 7.23 mmol) and N,N-diBoc-hydroxylamine (2.2 g, 9.4 mmol) inDMF (30 mL) at 0° C. under N₂. After 17 hours, the reaction mixture wasconcentrated. The residue was diluted with H₂O (50 mL) and extractedwith EtOAc (3×100 mL). The organic layers were combined, dried overanhydrous MgSO₄, filtered and concentrated. The crude product waspurified by column chromatography to produce the compound 22d (2.1 g,55%). ¹H-NMR (400 MHz, CDCl₃) δ 4.17 (s, 2H), 4.08 (t, J=5.2 Hz, 2H),3.78-3.60 (m, 21H), 1.53 (s, 18H).

Preparation of Compound 22e

To a solution of compound 22d (2.1 g, 3.99 mmol) in THF/MeOH/H₂O (30mL/10 mL/10 mL) was added NaOH (400 mg, 9.98 mmol) at 0° C. under N₂.The reaction mixture was stirred for 3 hours at room temperature. Thenthe pH of the solution was adjusted to 4-5 with 1 N aqueous HCl. Thereaction mixture was poured into H₂O (50 mL) and extracted with EtOAc(2×100 mL). The organic layers were combined, dried over MgSO₄.Filtration and concentration produced the compound 22e (1.6 g) ascolorless oil, which was used without further purification. ¹H-NMR (400MHz, CDCl₃) δ 7.90 (s, 1H), 4.15 (s, 2H), 4.03 (br s, 2H), 3.80-3.60 (m,18H), 1.47 (s, 9H).

Preparation of Compound 22f

DIPEA (0.13 mL, 0.73 mmol) and HBTU (187 mg, 0.49 mmol) were added to astirred solution of compound 21f (200 mg, 0.24 mmol) and compound 22e(152 mg, 0.36 mmol) in DMF (5 mL). The reaction mixture was stirred atroom temperature for 6 hours under N₂. The reaction mixture was dilutedH₂O (100 mL) and extracted with EtOAc (3×100 mL). The combined organiclayers were dried over anhydrous Na₂SO₄, filtered and concentrated underreduced pressure. The crude product was purified by columnchromatography to produce the compound 22f (100 mg, 34%). EI-MS m/z:1/2[M+H]⁺ 1205.6.

Preparation of Compound 22g

To a solution of compound 22f (100 mg, 0.08 mmol) in MeOH (20 mL) wasadded Pd/C (10 wt. %, 20 mg) and then the reaction mixture was stirredat room temperature for 4 hours under hydrogen. The reaction mixture wasfiltered through a celite pad and washed with MeOH (20 mL).Concentration provided compound 22g as colorless oil (70 mg), which wasused without further purification. EI-MS m/z: [M+H]⁺ 937.4, [M+Na]⁺959.3.

Preparation of Compound 22h

Compound 22h was prepared from compound 1i and compound 22g by a similarmethod of preparing compound 20q in Example 30. EI-MS m/z: 1/2[M+H]⁺1489.4.

Example 33. Preparation of Compound 23h

Preparation of Compound 23a

To a solution of compound 4a (483 mg, 0.69 mmol) in THF (10 mL) wasadded LiBr (180 mg, 2.06 mmol). The reaction mixture refluxed for 12hours under N₂. Then the reaction mixture was concentrated under reducedpressure. The residue was purified by column chromatography to producethe compound 23a (330 mg, 78%). ¹H-NMR (400 MHz, CDCl₃) δ 3.81 (t, J=6.4Hz, 2H), 3.72-3.59 (m, 44H), 3.47 (t, J=6.4 Hz, 2H).

Preparation of Compound 23b

To a solution of compound 23a (330 mg, 0.54 mmol) in acetone (2 mL) at0° C. was added Jones reagent (2 mL). After 15 hours at 0° C., thereaction mixture was filtered and concentrated. The residue was dilutedwith H₂O (15 mL) and extracted with EtOAc (2×20 mL). The organic layerswere combined, dried over anhydrous MgSO₄, filtered and concentrated.The resulting crude compound 23b was used without further purification.

Preparation of Compound 23c

To a solution of crude compound 23b (266 mg, 0.43 mmol) in MeOH (5 mL)was added oxalyl chloride (0.054 mL, 0.64 mmol) at 0° C. under N₂. After16 hours, the reaction mixture was concentrated and purified by columnchromatography, which produced the compound 23c (200 mg, 58% for 2steps). ¹H-NMR (400 MHz, CDCl₃) δ 4.17 (s, 2H), 3.81 (t, J=6.4 Hz, 2H),3.79-3.64 (m, 43H), 3.48 (t, J=6.4 Hz, 2H).

Preparation of Compound 23d

To a solution of compound 23c (200 mg, 0.31 mmol) in DMF (3 mL) wereadded N,N-diBoc-hydroxylamine (95 mg, 0.40 mmol) and NaH (60% in oil, 16mg, 0.37 mmol) at 0° C. under N₂. After 17 hours, the reaction mixturewas concentrated. The residue was diluted with H₂O (5 mL) and extractedwith EtOAc (3×10 mL). The organic layers were combined, dried overanhydrous MgSO₄, filtered and concentrated. The crude product waspurified by column chromatography to produce the compound 23d (120 mg,49%). ¹H-NMR (400 MHz, CDCl₃) δ 4.17 (s, 2H), 4.13 (t, J=8.0 Hz, 2H),3.75-3.64 (m, 45H), 1.53 (s, 18H).

Preparation of Compound 23e

To a solution of compound 23d (120 mg, 0.15 mmol) in THF/MeOH/H₂O (3mL/1 mL/1 mL) was added NaOH (15 mg, 0.38 mmol) at 0° C. under N₂. Thereaction mixture was stirred for 1 hour at room temperature. Then the pHof the solution was adjusted to 4-5 with 1 N aqueous HCl. The reactionmixture was poured into H₂O (10 mL) and extracted with CHCl₃ (2×20 mL).The organic layers were combined, dried over anhydrous Na₂SO₄.Filtration and concentration produced the compound 23e (100 mg), whichwas used without further purification. ¹H-NMR (400 MHz, CDCl₃) δ 4.23(t, J=8.0 Hz, 2H), 4.15 (s, 2H), 4.08 (t, J=4.0 Hz, 1H), 4.01 (t, J=4.0Hz, 1H), 3.74-3.64 (m, 40H), 1.53 (s, 9H).

Preparation of Compound 23f

DIPEA (0.052 mL, 0.29 mmol) and HBTU (75 mg, 0.20 mmol) were added to astirred solution of compound 21f (80 mg, 0.09 mmol) and compound 23e(100 mg, 0.15 mmol) in DMF (3 mL). After stirring at room temperaturefor 6 hours under N₂, the reaction mixture was diluted with H₂O (50 mL)and extracted with EtOAc (3×50 mL). The combined organic layers weredried over anhydrous Na₂SO₄, filtered and concentrated under reducedpressure. The crude product was purified by column chromatography toproduce the compound 23f (140 mg, 97%). ¹H-NMR (400 MHz, CDCl₃) δ7.38-7.31 (m, 10H), 5.44 (br, 2H), 5.09 (s, 4H), 4.34 (s, 2H), 4.26-4.17(m, 4H), 4.09-4.08 (m, 1H), 4.07 (br, 1H), 3.73-3.47 (m, 76H), 3.39-3.38(m, 4H), 1.53 (s, 9H). EI-MS m/z: [M+Na]⁺ 1491.6, [M+H-Boc]⁺: 1369.6.

Preparation of Compound 23g

To a solution of compound 23f (140 mg, 0.09 mmol) in MeOH (20 mL) wasadded Pd/C (10 wt. %, 20 mg) and then the reaction mixture was stirredat room temperature for 4 hours under hydrogen. The reaction mixture wasfiltered through a celite pad and washed with MeOH (20 mL).Concentration provided compound 23g as colorless oil (120 mg), which wasused without further purification. EI-MS m/z: [M+H]⁺ 1201.7, [M+Na]⁺1223.7.

Preparation of Compound 23h

Compound 23h was prepared from compound 1i and compound 23g by a similarmethod of preparing compound 20q in Example 30. EI-MS m/z: 1/2[M+H]⁺1620.3, 1/2[M+Na]⁺ 1632.1.

Example 34. Preparation of Compound 24l

Preparation of Compound 24a

Jones reagent (90 mL) was slowly added to a solution of compound2-[2-(2-chloroethoxy)ethoxy]ethanol (15.0 g, 88.9 mmol) in acetone (600mL) at 0° C. After 15 hours at 0° C., the reaction mixture was filteredand concentrated. The residue was diluted with H₂O (200 mL) andextracted with CHCl₃ (5×300 mL). The organic layers were combined, driedover anhydrous MgSO₄. Concentration provided compound 24a (20.0 g),which was used without further purification. ¹H-NMR (400 MHz, CDCl₃) δ4.18 (s, 2H), 3.81-3.64 (m, 8H).

Preparation of Compound 24b

To a solution of compound 24a (20.0 g, 88.9 mmol) in MeOH (500 mL) wasadded oxalyl chloride (11.5 mL, 133.4 mmol) at 0° C. for 30 minutesunder N₂. After 16 hours, the reaction mixture was concentrated andpurified by column chromatography, which produced the compound 24b (13.0g, 75%). ¹H-NMR (400 MHz, CDCl₃) δ 4.18 (s, 2H), 3.78-3.67 (m, 9H), 3.65(t, J=5.6 Hz, 2H).

Preparation of Compound 24c

Compound 24b (13.0 g, 66.1 mmol) and NaN₃ (6.4 g, 99.2 mmol) weredissolved in DMF (130 mL). After stirring at 100° C. for 2 hours, thereaction mixture was diluted with brine (200 mL) and extracted withCHCl₃ (2×100 mL). The organic layers were combined, dried over anhydrousMgSO₄. Concentration provided compound 24c (11.7 g, 87%), which was usedwithout further purification. ¹H-NMR (400 MHz, CDCl₃) δ 4.18 (s, 2H),3.76-3.67 (m, 9H), 3.41 (t, J=5.6 Hz, 2H).

Preparation of Compound 24d

To a solution of compound 24c (11.5 g, 56.6 mmol) in THF/MeOH/H₂O (300mL/100 mL/100 mL) was added NaOH (4.53 g, 113.2 mmol) at 0° C. After 2hours at 0° C. under N₂, the pH of the solution was adjusted to 2 with 4M aqueous HCl. The reaction mixture was poured into H₂O (100 mL) andextracted with CHCl₃ (3×500 mL). The organic layers were combined, driedover MgSO₄. Filtration and concentration produced the compound 24d (10.7g, 99%) as colorless oil, which was used without further purification.¹H-NMR (400 MHz, CDCl₃) δ 4.19 (s, 2H), 3.79-3.77 (m, 2H), 3.72-3.70 (m,4H), 3.44 (t, J=5.2 Hz, 2H).

Preparation of Compound 24e

A three-necked flask was loaded consecutively with H₂O (40 mL),1,4-dioxane (70 mL) and H-Lys(Z)—OH (10 g, 35.7 mmol). The mixture wasstirred until complete dissolution. The pH was adjusted to about 10.5 byadding of 2 M aqueous Na₂CO₃. Benzyl chloroformate (6.69 g, 39.2 mmol)was added while maintaining the pH at about 10-11 by adding at the sametime 2 M aqueous Na₂CO₃. After completing addition, the reaction mixturewas stirred at 20° C. for 1 hour. Then EtOAc (50 mL) was added and pH ofthe resulting mixture was adjusted to 2-3 with c-HCl. The organic layerwas separated and the aqueous layer was extracted with EtOAc (50 mL).The combined organic layers were washed with brine (50 mL), and driedover Na₂SO₄. Filtration and concentration under reduced pressure yieldedthe compound 24e as yellowish oil (14.7 g, 99%). ¹H-NMR (400 MHz, CDCl₃)δ 7.33-7.27 (m, 10H), 5.07-5.04 (d, 4H), 4.08 (m, 1H), 3.09 (t, 2H),1.51 (br s, 1H), 1.49 (bs, 1H), 1.47-1.40 (m, 4H).

Preparation of Compound 24f

DIPEA (0.40 mL, 2.37 mmol), HOBt (143 mg, 1.06 mmol) and EDC.HCl (240mg, 1.25 mmol) were added to a stirred mixture of compound 24e (400 mg,0.96 mmol) and compound 2d (261 mg, 0.86 mmol) in DMF (3 mL). Afterstirring at room temperature for 14 hours under N₂, the reaction mixturewas poured into H₂O (50 mL), extracted with EtOAc (3×50 mL), washed withaq NaHCO₃ (50 mL) and brine (50 mL) and dried over anhydrous Na₂SO₄.After filtration and concentration under reduced pressure, the resultingresidue was purified by column chromatography to yield the compound 24f(380 mg, 59%). ¹H NMR (400 MHz, CDCl₃) δ 8.16 (s, 1H), 7.34-7.28 (m,10H), 7.49 (s, 1H) 5.08-5.07 (m, 5H), 4.17 (m, 1H), 3.99 (t, 2H),3.68-3.16 (m, 10H), 3.17 (d, 2H), 1.66 (m, 1H), 1.51-1.27 (m, 14H).EI-MS m/z: [M+H]⁺ 661.0.

Preparation of Compound 24g

To a stirred mixture of compound 24f (370 mg, 0.55 mmol) and Pd/C (10wt. %, 74 mg) in MeOH (10 mL) at 0° C. was added HCl (4 N in1,4-dioxane, 0.27 mL, 1.1 mmol). After stirring at room temperature for2 h under hydrogen, the reaction mixture was filtered through a celitepad and washed with MeOH (40 mL). The filtrate was concentrated toproduce the compound 24g (223 mg, 87%) as colorless oil, which was usedwithout further purification. ¹H-NMR (400 MHz, DMSO-d₆) δ 10.02 (s, 1H),8.62 (s, 1H), 8.22 (br, 2H), 7.90 (br, 2H), 3.81 (t, 2H), 3.56 (m, 4H),3.46 (t, 2H), 3.39-3.27 (m, 26H), 2.75 (m, 2H), 1.73 (q, 2H), 1.55 (p,2H), 1.40-1.33 (m, 14H).

Preparation of Compound 24h

DIPEA (1.6 mL, 9.45 mmol), HOBt (746 mg, 5.52 mmol) and EDC.HCl (1.19 g,6.42 mmol) were added to a stirred mixture of compound 24g (1.0 g, 5.29mmol) and compound 24d (1.1 g, 2.35 mmol) in DMF (15 mL). After stirringat room temperature for 14 hours under N₂, the reaction mixture waspoured into H₂O (20 mL), extracted with DCM (3×50 mL) and dried overanhydrous Na₂SO₄. After filtration and concentration under reducedpressure, the resulting residue was purified by column chromatography toyield the compound 24h (1.25 g, 70%). ¹H-NMR (400 MHz, CDCl₃) δ 8.36 (s,1H), 7.30 (d, 1H), 7.08 (s, 1H), 7.68 (t, 1H), 4.46 (q, 1H),4.07-3.98-4.01 (m, 4H), 3.98 (s, 2H), 3.75-3.663 (m, H) 3.57 (t, 2H),3.44 (m, 6H), 3.28 (m, 2H), 1.87 (m, 1H), 1.66 (m, 1H), 1.59-1.52 (p,2H), 1.48 (s, 9H), 1.41-1.33 (m, 2H). EI-MS m/z: [M+H]⁺ 735.0.

Preparation of Compound 24i

To a stirred mixture of compound 24h (1.2 g, 0.163 mmol), and Pd/C (10wt. %, 250 mg) in MeOH (30 mL) at 0° C., 4 N HCl (1,4-dioxane, 0.81 mL,3.26 mmol) was added. After stirring at room temperature for 1.5 hoursunder hydrogen, the reaction mixture was filtered through a celite padand washed with MeOH (100 mL). The filtrate was concentrated to producethe compound 24i (1.39 g, 99%) as colorless oil, which was used withoutfurther purification. ¹H-NMR (400 MHz, DMSO-d₆) δ 9.99 (s, 1H), 8.22 (t1H) 7.74 (t, 1H), 7.61 (d, 1H), 4.31, (q, 1H), 3.93 (s, 2H), 3.86 (s,2H), 3.79 (t, 2H), 3.60-3.50 (m, 18H), 3.06 (q, 2H), 2.97 (p, 4H),1.60-1.49 (m, 2H), 1.39 (m, 11H), 1.20 (m, 2H). EI-MS m/z: [M+H]⁺ 683.

Preparation of Compound 24j

DIPEA (0.021 mL, 0.125 mmol) and HBTU (29 mg, 0.078 mmol) were added toa stirred mixture of compound 1i (85 mg, 0.069 mmol) and compound 24i(23 mg, 0.031 mmol) in DMF (0.7 mL). After stirring at room temperaturefor 14 hours under N₂, the reaction mixture was dissolved in H₂O/DMSO(1.5 mL/1.5 mL) and purified by HPLC. Pure fractions with the sameretention time were combined and concentrated to produce the compound24j (67 mg, 68%). EI-MS m/z: 1/2[M+H]⁺ 1552.5.

Preparation of Compound 24k

To a solution of compound 24j (67 mg, 0.021 mmol) in MeOH (1.7 mL) wasadded LiOH monohydrate (16 mg, 0.388 mmol) in H₂O (1.7 mL) at 0° C.After stirring for 2 hours at 0° C., the reaction mixture wasneutralized using acetic acid (0.018 mL) and concentrated under reducedpressure. The reaction mixture was dissolved in H₂O/DMSO (1.5 mL/1.5 mL)and purified by HPLC. Pure fractions with the same retention time werecombined and concentrated to produce the compound 24k (37 mg, 62%).EI-MS m/z: 1/2[M+H]⁺ 1412.3.

Preparation of Compound 24l

TFA (0.4 mL) was added to a stirred solution of compound 24k (37 mg,0.013 mmol) in DCM (2.0 mL). After stirring at 0° C. for 2 hours, thesolvent and excess TFA were blown off with N₂. The residue was dissolvedin H₂O/acetonitrile (1 mL/1 mL) and purified by HPLC. Pure fractionswith the same retention time were combined and lyophilized to producethe compound 24l (19.8 mg, 53%) as white solid. EI-MS m/z: 1/2[M+H]⁺1362.3.

Example 35. Preparation of Compound 25e

Preparation of Compound 25a

Compound 22c (1.0 g, 2.67 mmol) and NaN₃ (261 mg, 4.01 mmol) weredissolved in DMF (3 mL). The reaction mixture was heated at 100° C. for5 hours. After the reaction was completed, the reaction mixture wasfiltered and concentrated. The residue was purified by columnchromatography (EtOAc to EtOAc/MeOH 10/1), which produced the compound25a (854 mg, 95%).

¹H-NMR (400 MHz, CDCl₃) δ 4.17 (s, 2H), 3.76-3.64 (m, 21H), 3.39 (t,J=5.2 Hz, 2H).

Preparation of Compound 25b

To a stirred solution of compound 25a (854 mg, 2.54 mmol) in MeOH (25mL) at 0° C., 2 M aq. NaOH (6.3 mL, 12.64 mmol) was added. The reactionmixture was stirred at room temperature for 3 hours. The solution wasthen concentrated under reduced pressure. The resulting suspension wasacidified with aqueous 2 N HCl while cooling at 0° C. The residue wasextracted by CHCl₃ (8×500 mL). The organic layers were combined, driedover Na₂SO₄ and concentrated to produce the compound 25b (783 mg, 96%).¹H-NMR (400 MHz, CDCl₃) δ 4.16 (s, 2H), 3.76-3.65 (m, 18H), 3.40 (t,J=5.2 Hz, 2H).

Preparation of Compound 25c

DIPEA (0.30 mL, 1.70 mmol) and HBTU (483 mg, 1.27 mmol) were added to astirred mixture of compound 25b (337 mg, 1.05 mmol) and compound 24g(198 mg, 0.42 mmol) in DMF (3 mL). After stirring at room temperaturefor 14 hours under N₂, the reaction mixture was concentrated andpurified by column chromatography (EtOAc to EtOAc/MeOH 10/1), whichproduced the compound 25c (358 mg, 84%). ¹H-NMR (400 MHz, DMSO-d₆) δ9.98 (s, 1H), 8.09 (t, J=5.2 Hz, 1H), 7.63 (t, J=5.2 Hz, 1H), 7.55 (d,J=8.4 Hz, 1H), 4.31-4.25 (m, 1H), 3.90 (s, 2H), 3.84 (s, 2H), 3.80 (m,2H), 3.62-3.46 (m, 34H), 3.42-3.36 (m, 6H), 3.25-3.17 (m, 2H), 3.08-3.03(m, 2H), 1.61-1.51 (m, 2H), 1.39 (s, 9H), 1.26-1.10 (m, 7H). EI-MS m/z:[M+H]⁺ 999.1.

Preparation of Compound 25d

To a solution of compound 25c (358 mg, 0.35 mmol) in MeOH (7 mL) wasadded Pd/C (10 wt. %, 38 mg) and HCl (4 N in 1,4-dioxane, 0.18 mL, 0.72mmol). After stirring at room temperature for 5 hours under hydrogen,the reaction mixture was filtered through a celite pad and washed withMeOH (400 mL). The filtrate was concentrated to produce the compound 25d(314 mg, 93%) as colorless oil, which was used without furtherpurification. ¹H-NMR (400 MHz, DMSO-d₆) δ 9.98 (s, 1H), 8.10 (m, 1H),7.68 (m, 1H), 7.57 (m, 1H), 4.31-4.25 (m, 1H), 3.90 (s, 2H), 3.84 (s,2H), 3.80 (m, 2H), 3.62-3.46 (m, 30H), 3.42-3.36 (m, 10H), 3.45-3.16 (m,4H), 3.08-3.03 (m, 3H), 2.72-2.66 (m, 3H), 1.61-1.51 (m, 2H), 1.39 (s,9H), 1.26-1.10 (m, 6H). EI-MS m/z: [M+H]⁺ 947.1.

Preparation of Compound 25e

Compound 25e was prepared from compound 1i and compound 25d by a similarmethod of preparing compound 24l in Example 34. EI-MS m/z: 1/2[M+H]⁺1493.7.

Example 36. Preparation of Compound 25f

Compound 25f was prepared from compound 1j and compound 25d by a similarmethod of preparing compound 24l in Example 34. EI-MS m/z: 1/2[M+H]⁺1508.2.

Example 37. Preparation of Compound 26e

Preparation of Compound 26a

DIPEA (0.65 mL, 0.004 mmol), HOBt (218 mg, 1.61 mmol) and EDC.HCl (364mg, 1.9 mmol) were added to a stirred mixture of compound 24e (1.0 g,2.43 mmol) and compound 3e (810 mg, 1.52 mmol) in DMF (10 mL). Afterstirring at room temperature for 14 hours under N₂, the reaction mixturewas poured into H₂O (50 mL) and extracted with EtOAc (3×50 mL). Thecombined organic layers were washed with 1 N aq. HCl (30 mL), saturatedaq. NaHCO₃ (30 mL), and brine (30 mL), and dried over anhydrous Na₂SO₄.After filtration and concentration, the residue was purified by columnchromatography, which produced the compound 26a (988 mg, 73%).

¹H-NMR (400 MHz, CDCl₃) δ 7.33-7.26 (m, 8H), 6.85 (s, 1H), 5.63 (s, 1H),5.08-5.02 (s, 4H), 4.16-4.11 (m, 1H), 4.09-4.05 (m, 2H), 3.72-3.70 (m,2H), 3.62-3.59 (m, 14H), 3.53 (s, 2H), 3.44-3.43 (m, 2H), 3.18-3.16 (m,2H), 1.82 (m, 1H), 1.72 (s, 7H), 1.66 (m, 1H), 1.52 (s, 18H), 1.38-1.36(m, 2H), 1.24-1.27 (s, 1H). EI-MS m/z: [M+H−2Boc]⁺ 693.1.

Preparation of Compound 26b

To a stirred mixture of compound 26a (988 mg, 1.1 mmol) and Pd/C (10 wt.%, 196 mg) in MeOH (6 mL) at 0° C. was added HCl (4 N in 1,4-dioxane,0.55 mL, 2.2 mmol). After stirring at room temperature for 1.5 hoursunder hydrogen, the reaction mixture was filtered through a celite padand washed with MeOH (40 mL). The filtrate was concentrated to producethe compound 26b (767 mg, 99%) as a yellow form, which was used withoutfurther purification. EI-MS m/z: [M+H]⁺ 625.0, [M+H-Boc]⁺ 525.0,[M+H−2Boc]⁺ 424.9.

Preparation of Compound 26c

DIPEA (0.2 mL, 1.14 mmol), HOBt (89 mg, 0.66 mmol) and EDC.HCl (142 mg,0.74 mmol) were added to a stirred mixture of compound 26b (200 mg, 0.29mmol) and compound 25b (202 mg, 0.63 mmol) in DMF (5 mL). After stirringat room temperature for 14 hours under N₂, the reaction mixture waspoured into H₂O (10 mL) and extracted with DCM (3×10 mL). The combinedorganic layers were dried over anhydrous Na₂SO₄. After filtration andconcentration, the residue was purified by column chromatography, whichproduced the compound 26c (270 mg, 77%). ¹H-NMR (400 MHz, CDCl₃) δ 8.07(t, 1H), 7.62 (t, 1H), 7.54-7.52 (m, 1H), 5.73 (s, 2H), 4.27-4.25 (q,1H), 3.96 (t, 2H), 3.88 (s, 2H), 3.82 (s, 2H), 3.58-3.48 (m, 52H),3.19-3.18 (m, 3H), 3.04-3.03 (m, 3H), 1.44 (s, 18H), 1.39-1.37 (m, 3H),1.21-1.19 (m, 3H). EI-MS m/z: [M+H−2Boc]⁺ 1031.6.

Preparation of Compound 26d

To a stirred mixture of compound 26c (160 mg, 0.13 mmol) and Pd/C (10wt. %, 28 mg) in MeOH (20 mL) at 0° C. was added HCl (4 N in1,4-dioxane, 0.07 mL, 0.28 mmol). After stirring at room temperature for30 minutes under hydrogen, the reaction mixture was filtered through acelite pad and washed with MeOH (30 mL). The filtrate was concentratedto produce the compound 26d (140 mg, 91%) as colorless oil, which wasused without further purification. EI-MS m/z: [M+H]⁺ 1179.7.

Preparation of Compound 26e

Compound 26e was prepared from compound 1i and compound 26d by a similarmethod of preparing compound 24l in Example 34. EI-MS m/z: 1/2[M+H]⁺1560.6, 1/3[M+H]⁺ 1040.7.

Example 38. Preparation of Compound 27e

Preparation of Compound 27a

DIPEA (0.19 ml, 1.1 mmol), HOBt (64 mg, 0.47 mmol), and EDC.HCl (91 mg,0.47 mmol) were added to a stirred mixture of compound 24e (228 mg, 0.55mmol) and compound 4e (256 mg, 0.36 mmol) in DMF (4 mL). After stirringat room temperature for 4 hours under N₂, the reaction mixture waspoured into H₂O (10 mL) and extracted with EtOAc (3×15 mL). The combinedorganic layers were washed with 1 N aq. HCl (20 mL), saturated aq.NaHCO₃ (10 mL), and brine (10 mL), and dried over anhydrous Na₂SO₄.After filtration and concentration, the residue was purified by columnchromatography, which produced the compound 27a (327 mg, 85%).

¹H-NMR (400 MHz, CDCl₃) δ 7.73 (s, 1H), 7.33-7.26 (m, 11H), 6.91 (s,1H), 5.67 (br, 1H) 5.08-5.07 (m, 5H), 4.15 (m, 1H), 4.02 (t, 2H),3.72-3.44 (m, 46H), 3.16 (d, 2H), 1.82 (m, 1H), 1.63 (m, 1H), 1.55-1.36(m, 13H).

Preparation of Compound 27b

To a stirred mixture of compound 27a (327 mg, 0.309 mmol) and Pd/C (10wt. %, 65 mg) in MeOH (6 mL) at 0° C. was added HCl (4 N in 1,4-dioxane,0.15 mL, 0.618 mmol). After stirring at room temperature for 1.5 hoursunder hydrogen, the reaction mixture was filtered through a celite padand washed with MeOH (40 mL). The filtrate was concentrated to producethe compound 27b (244 mg, 91%) as colorless oil, which was used withoutfurther purification. EI-MS m/z: [M+H]⁺ 789.2.

Preparation of Compound 27c

DIPEA (0.19 ml, 1.13 mmol), HOBt (95 mg, 0.707 mmol), and EDC.HCl (135mg, 0.707 mmol) were added to a stirred mixture of compound 25b (227 mg,0.707 mmol) and compound 27b (244 mg, 0.283 mmol) in DMF (6 mL). Afterstirring at room temperature for 3 hours under N₂, the reaction mixturewas poured into H₂O (5 mL) and extracted with DCM (3×10 mL). Thecombined organic layers were dried over anhydrous Na₂SO₄. Afterfiltration and concentration, the residue was purified by columnchromatography, which produced the compound 27c (339 mg, 85%). ¹H NMR(400 MHz, CDCl₃) δ 7.69 (s, 1H), 7.29 (d, 1H), 6.99 (s, 1H), 6.82 (s,1H), 4.39 (q, 1H), 3.99-3.94 (m, 6H), 3.69-3.58 (m, 80H), 3.51 (t, 2H),3.44-3.34 (m, 8H), 3.25 (m, 2H), 1.68-1.64 (m, 1H). 1.53-1.48 (m, 2H),1.44 (s, 9H), 1.33 (m, 2H). EI-MS m/z: [M+H]⁺ 1395.6.

Preparation of Compound 27d

To a stirred mixture of compound 27c (339 mg, 0.242 mmol), and Pd/C (10wt. %, 67 mg) in MeOH (6 mL) at 0° C. was added HCl (4 N in 1,4-dioxane,0.12 mL, 0.484 mmol). After stirring at room temperature for 30 minutesunder hydrogen, the reaction mixture was filtered through a celite padand washed with MeOH (30 mL). The filtrate was concentrated to producethe compound 27d (300 mg, 87%) as colorless oil, which was used withoutfurther purification. EI-MS m/z: [M+H]⁺ 1343.5.

Preparation of Compound 27e

Compound 27e was prepared from compound 1i and compound 27d by a similarmethod of preparing compound 24l in Example 34. EI-MS m/z: 1/2[M+H]⁺1692.5.

Example 39. Preparation of Compound 28d

Preparation of Compound 28c

Compound 28c was prepared from H-D-Lys(Z)—OH by a similar method ofpreparing compound 25d in Example 35.

Preparation of Compound 28d

Compound 28d was prepared from compound 1i and compound 28c by a similarmethod of preparing compound 25e in Example 35. EI-MS m/z: 1/2[M+H]⁺1494.9.

Example 40. Preparation of Compound 28e

Compound 28e was prepared from compound 1j and compound 28c by a similarmethod of preparing compound 25e in Example 35. EI-MS m/z: 1/2[M+H]⁺1509.2.

Example 41. Preparation of Compound 29j

Preparation of Compound 29a

To a solution of hexaethylene glycol (25.0 g, 88.5 mmol) in DCM (100 mL)were added triethylamine (61.7 mL, 443 mmol) and p-toluenesulfonylchloride (50.6 g, 266 mmol) at 0° C. under N₂. After 5 hours at 0° C.,the reaction mixture was poured into 1 N aq. HCl (200 mL) and extractedwith DCM (2×200 mL). The combined organic layers were washed withsaturated aq. NaHCO₃ (100 mL) and brine (100 mL), and dried overanhydrous Na₂SO₄. After filtration and concentration, the residue waspurified by column chromatography, which produced the compound 29a (45.0g, 87%) as brown oil. ¹H-NMR (400 MHz, CDCl₃) δ 7.79 (d, J=7.6 Hz, 4H),7.34 (d, J=7.6 Hz, 4H), 4.16-4.14 (m, 4H), 3.69-3.67 (m, 4H), 3.64-3.56(m, 16H), 2.44 (s, 6H).

Preparation of Compound 29b

To a solution of compound 29a (17.6 g, 29.7 mmol) in DMF (100 mL) wereadded NaN₃ (9.65 g, 148 mmol) and tetrabutylammonium iodide (550 mg,1.49 mmol). The reaction mixture was heated up to 80° C. After stirringfor 16 hours at 80° C., the reaction mixture was allowed to cool to roomtemperature. The reaction mixture was filtered through a celite pad andwashed with DCM (100 mL). After concentration, the residue was purifiedby column chromatography, which produced the compound 29b (9.4 g, 94%).¹H-NMR (400 MHz, CDCl₃) δ 3.68 (m, 20H), 3.39 (t, 4H).

Preparation of Compound 29c

To a solution of 29b (8.4 g, 24.9 mmol) in DCM (24 mL) and toluene (24mL) were added 1 N aq. HCl (40.3 mL) and triphenylphosphine (6.9 g, 23.6mmol). The reaction mixture was stirred at room temperature under N₂ for16 hours. After removal of the solvent under reduced pressure, H₂O (20mL) was added into the reaction mixture, and the aqueous layer wasextracted with EtOAc (20 mL). Then the pH of the aqueous phase wasadjusted to 13. The resulting aqueous phase was extracted with DCM (3×30mL). The combined organic layers were dried over anhydrous Na₂SO₄,filtered, and concentrated under reduced pressure to produce thecompound 29c (6.6 g, 84%) as colorless oil. ¹H-NMR (400 MHz, CDCl₃) δ3.67 (m, 20H), 3.52 (t, 2H), 3.39 (t, 2H), 2.86 (t, 2H). EI-MS m/z:[M+H]⁺ 306.9.

Preparation of Compound 29d

DIPEA (2.67 mL, 15.4 mmol) and HBTU (3.49 g, 9.21 mmol) were added to astirred mixture of L-glutamic acid dimethyl ester hydrochloride (1.3 g,6.14 mmol) and compound 20e (1.72 g, 6.14 mmol) in DMF (15 mL) at 0° C.The reaction mixture was stirred at 0° C. for 30 minutes and allowed towarm to room temperature over 16 hours under N₂. The reaction mixturewas poured into water (50 mL) and extracted with DCM (3×50 mL). Thecombined organic layers were washed with 0.5 N HCl (50 mL), saturatedaq. NaHCO₃ (50 mL) and brine (50 mL) sequentially, and dried overanhydrous Na₂SO₄. After filtration and concentration under reducedpressure, the resulting crude product was purified by columnchromatography to produce the compound 29d (2.18 g, 81%). ¹H-NMR (400MHz, DMSO-d₆) δ 10.00 (s, 1H), 8.04 (d, 1H), 4.34 (m, 1H), 3.93 (s, 1H),3.77 (s, 1H), 3.63 (s, 3H), 3.58 (s, 9H), 3.38-3.34 (t, 2H), 2.14 (m,H), 1.90 (m, 1H), 1.39 (s, 9H). EI-MS m/z: [M+H]⁺ 437.35.

Preparation of Compound 29e

To a solution of compound 29d (2.18 g, 4.99 mmol) in THF:MeOH:H₂O (12mL:4 mL:4 mL) was added NaOH (499 mg, 12.5 mmol) at room temperatureunder N₂. After 3 hours, the pH of the reaction mixture was adjusted to4 and concentrated. Then the residue was extracted with DCM/MeOH (80mL/20 mL). Concentration provided compound 29e (1.0 g, 49%) as yellowoil, which was used without further purification. EI-MS m/z: [M+H-Boc]⁺309.20.

Preparation of Compound 29f

DIPEA (1.7 mL, 9.79 mmol) and HBTU (2.79 g, 7.35 mmol) were added to astirred mixture of compound 29e (1.0 g, 2.45 mmol) and compound 29c(2.25 g, 7.35 mmol) in DMF (10 mL) at 0° C. The reaction mixture wasstirred at 0° C. for 30 minutes and allowed to warm to room temperatureover 16 hours under N₂. The reaction mixture was poured into water (50mL) and extracted with DCM (3×50 mL). The combined organic layers werewashed with 0.5 N aq. HCl (50 mL), saturated aq. NaHCO₃ (50 mL) andbrine (50 mL) sequentially, and dried over anhydrous Na₂SO₄. Afterfiltration and concentration under reduced pressure, the resulting crudeproduct was purified by column chromatography to produce the compound29f (611 mg, 25%). ¹H-NMR (400 MHz, DMSO-d₆) δ 9.97 (s, 1H), 8.08 (t,1H), 7.85 (t, 1H), 7.64 (d, 1H), 4.27 (m, 1H), 3.83 (s, 2H), 3.82-3.61(m, 2H), 3.61-3.50 (m, 42H), 3.42-3.37 (m, 8H), 3.28-3.15 (m, 4H), 2.90(s, H), 2.08-2.04 (m, 2H), 1.88 (m, 1H), 1.75 (m, 1H), 1.39 (s, 9H).EI-MS m/z: [M+H]⁺ 986.73.

Preparation of Compound 29g

To a stirred mixture of compound 29f (611 mg, 0.62 mmol) in MeOH (50 mL)was added Pd/C (10 wt. %, 132 mg 0.62 mmol). After stirring at roomtemperature for 2 hours under hydrogen, the reaction mixture wasfiltered through a celite pad and washed with MeOH (40 mL). The filtratewas concentrated to produce the compound 29g as colorless oil (518 mg,crude), which was used without further purification. EI-MS m/z: [M+H]⁺933.85.

Preparation of Compound 29h

DIPEA (0.026 mL, 0.150 mmol) and HBTU (40 mg, 0.105 mmol) were added toa stirred mixture of compound 29g (35 mg, 0.037 mmol) and compound 1i(106 mg, 0.086 mmol) in DMF (3 mL). After stirring at room temperaturefor 16 hours under N₂, the reaction mixture was diluted water (20 mL)and extracted with EtOAc (3×10 mL). The combined organic layers werewashed with 0.5 N HCl (20 mL), saturated aq. NaHCO₃ (20 mL) and brine(20 mL) sequentially, and dried over anhydrous Na₂SO₄. After filtrationand concentration under reduced pressure, the resulting crude productwas purified by column chromatography to produce the compound 29h (81.4mg, 65%). EI-MS m/z: 1/2[M+H]⁺ 1677.94, 1/3[M+H]⁺ 1119.03.

Preparation of Compound 29i

To a solution of compound 29h (81 mg, 0.024 mmol) in MeOH (1 mL) wasadded LiOH monohydrate (8.1 mg, 0.19 mmol) in H₂O (1 mL) at −10° C.After stirring for 2 hours at −10° C., the reaction mixture wasneutralized using acetic acid and concentrated under reduced pressure.Then the reaction mixture was dissolved in H₂O/DMSO (1.5 mL/1.5 mL) andpurified by HPLC, which produced the compound 29i (53 mg, 72%) as whitesolid. EI-MS m/z: 1/2[M+H]⁺ 1537.86, 1/3[M+H]⁺ 1025.66.

Preparation of Compound 29j

TFA (0.3 mL) was added to a stirred solution of compound 29i (53 mg,0.017 mmol) in DCM (1.0 mL) at 0° C. After stirring for 1 hour, thesolvent and excess TFA were removed by N₂ flow. Then the residue wasdissolved in H₂O/MeCN (1 mL/1 mL) and purified by HPLC. Pure fractionswith the same retention time were combined and lyophilized to producethe compound 29j (23.1 mg, 43%) as white solid. EI-MS m/z: 1/2[M+H]⁺1487.99, 1/3 [M+H]⁺ 992.40.

Example 42. Preparation of Compound 29k

Compound 29k was prepared from compound 1j and compound 29g by a similarmethod of preparing compound 29j in Example 41. EI-MS m/z: 1/2[M+H]⁺1501.93, 1/3[M+H]⁺ 1001.69.

Example 43. Preparation of Compound 30b

Preparation of Compound 30a

Compound 30a was prepared from D-glutamic acid dimethyl esterhydrochloride by a similar method of preparing compound 29g in Example41. EI-MS m/z: [M+H]⁺ 933.89.

Preparation of Compound 30b

Compound 30b was prepared from compound 1i and compound 30a by a similarmethod of preparing compound 29j in Example 41. EI-MS m/z: 1/2[M+H]⁺1488.07, 1/3[M+H]⁺ 992.40.

Example 44. Preparation of Compound 30c

Compound 30c was prepared from compound 1j and compound 30a by a similarmethod of preparing compound 29j in Example 41. EI-MS m/z: 1/2[M+H]⁺1501.93, 1/3[M+H]⁺ 1001.69.

Example 45. Preparation of Compound 31f

Preparation of Compound 31a

A three-necked flask was loaded consecutively with H₂O (18 mL),1,4-dioxane (30 mL) and L-ornithine monohydrochloride (3.0 g, 17.8mmol). The mixture was stirred until complete dissolution. The pH wasadjusted to about 10.5 by addition of 2 M aq. Na₂CO₃. Benzylchloroformate (6.37 g, 37.4 mmol) was added while maintaining the pH atabout 10-11 by adding at the same time 2 M aq. Na₂CO₃. After the end ofthe addition, the reaction mixture was stirred at 20° C. for 1 hour.Then EtOAc (50 mL) was added and pH of the resulting mixture wasadjusted to 2-3 with c-HCl. The organic layer was separated and theaqueous layer was extracted with EtOAc (50 mL). The combined organiclayers were washed with brine (50 mL), and dried over Na₂SO₄. Filtrationand concentration under reduced pressure provided compound 31a (7.1 g).¹H-NMR (400 MHz, DMSO-d₆) δ 12.54 (s, 1H), 7.54 (s, 1H), 7.44-7.29 (m,10H), 7.24-7.22 (m, 1H), 5.16-5.00 (d, 4H), 3.95-3.89 (m, 1H), 3.00-2.96(m, 2H), 1.98-1.57 (m, 1H), 1.56-1.46 (m, 3H).

Preparation of Compound 31b

DIPEA (1.41 mL, 8.12 mmol) and HBTU (1.85 g, 4.87 mmol) were added to astirred mixture of compound 31a (1.30 g, 3.25 mmol) and compound 2d (891mg, 3.57 mmol) in DMF (10 mL) at 0° C. The reaction mixture was stirredat 0° C. for 30 minutes and allowed to warm to room temperature over 16hours under N₂. The reaction mixture was poured into water (50 mL) andextracted with DCM (3×50 mL). The combined organic layers were washedwith 0.5 N aq. HCl (50 mL), saturated aq. NaHCO₃ (50 mL) and brine (50mL) sequentially, and dried over anhydrous Na₂SO₄. After filtration andconcentration under reduced pressure, the resulting crude product waspurified by column chromatography to produce the compound 31b (1.2 g,57%). EI-MS m/z: [M+H]⁺ 647.54, [M+H-Boc]⁺ 547.47

Preparation of Compound 31c

To a stirred mixture of compound 31b (1.2 g, 1.86 mmol) in MeOH (50 mL)was added Pd/C (10 wt. %, 59 mg 5.57 mmol). After stirring at roomtemperature for 2 hours under hydrogen, the reaction mixture wasfiltered through a celite pad and washed with MeOH (40 mL). The filtratewas concentrated to produce the compound 31c (717 mg), which was usedwithout further purification. ¹H-NMR (400 MHz, DMSO-d₆) δ 7.93 (s, 1H),3.81 (t, 2H), 3.55 (t, 2H), 3.51 (s, 5H), 3.42-3.22 (m, 13H), 1.37 (s,9H).

Preparation of Compound 31d

DIPEA (0.55 mL, 3.17 mmol) and HBTU (902 mg, 2.38 mmol) were added to astirred mixture of compound 31c (300 mg, 0.79 mmol) and compound 25b(637 mg, 1.98 mmol) in DMF (5 mL) at 0° C. The reaction mixture wasstirred at 0° C. for 30 minutes and allowed to warm to room temperatureover 16 hours under N₂. The reaction mixture was poured into water (30mL) and extracted with DCM (3×30 mL). The combined organic layers werewashed with 0.5 N aq. HCl (30 mL), saturated aq. NaHCO₃ (30 mL) andbrine (30 mL) sequentially, and dried over anhydrous Na₂SO₄. Afterfiltration and concentration under reduced pressure, the resulting crudeproduct was purified by column chromatography to produce the compound31d (551 mg, 71%). EI-MS m/z: [M+H]⁺ 985.87.

Preparation of Compound 31e

To a stirred mixture of compound 31d (491 mg, 0.50 mmol) in MeOH (30 mL)was added Pd/C (10 wt. %, 106 mg 1.00 mmol). After stirring at roomtemperature for 2 hours under hydrogen, the reaction mixture wasfiltered through a celite pad and washed with MeOH (40 mL). The filtratewas concentrated to produce the compound 31e (452 mg), which was usedwithout further purification. EI-MS m/z: [M+H]⁺ 933.94.

Preparation of Compound 31f

Compound 31f was prepared from compound 1i and compound 31e by a similarmethod of preparing compound 25e in Example 35. EI-MS m/z: 1/2[M+H]⁺1488.20, 1/3[M+H]⁺ 992.54.

Example 46. Preparation of Compound 31g

Compound 31g was prepared from compound 1j and compound 31e by a similarmethod of preparing compound 25e in Example 35. EI-MS m/z: 1/2[M+H]⁺1502.23, 1/3[M+H]⁺ 1001.86.

Example 47. Preparation of Compound 32c

Preparation of Compound 32a

DIPEA (0.6 mL, 7.07 mmol) and HBTU (972 mg, 5.30 mmol) were added to astirring mixture of compound 24g (483 mg, 0.855 mmol) andFmoc-NH-PEGS-CH₂CH₂COOH (1.0 g, 3.89 mmol) in DMF (10 mL). Afterstirring at room temperature for 14 hours under N₂, the reaction mixturewas poured into H₂O (30 mL) and extracted with EtOAc (3×30 mL). Thecombined organic layers were washed with 1 N aq. HCl (10 mL), saturatedaq. NaHCO₃ (10 mL) and brine (10 mL) sequentially, and dried overanhydrous Na₂SO₄. After filtration and concentration, the resultingresidue was purified by column chromatography, which produced thecompound 32a (1.16 g, 90%). ¹H-NMR (400 MHz, CDCl₃) δ 7.77 (d, 4H), 7.60(d, 4H), 7.39 (t, 4H), 7.31 (t, 4H), 4.39 (d, 4H), 4.33 (m, 1H), 4.22(m, 2H), 4.09 (m, 2H), 3.71-3.39 (m, 52H), 3.19 (m, 2H), 2.51 (m, 4H),1.50 (m, 1H), 1.46 (m, 1H), 1.43 (s, 9H), 1.25 (m, 2H). EI-MS m/z:[M+H]⁺ 1520.0.

Preparation of Compound 32b

To a solution of compound 32a (500 mg, 0.328 mmol) in THF (8 mL) wasadded piperidine (2 mL) at room temperature. After stirring for 20minutes, the reaction mixture was concentrated under reduced pressure.The resulting residue was purified by column chromatography to producethe compound 32b (175 mg, 50%). ¹H-NMR (400 MHz, CDCl₃) δ 4.41 (m, 1H),4.01 (m, 2H), 3.75-3.56 (m, 43H), 3.54 (m, 2H), 3.24 (m, 2H), 2.89 (m,3H), 2.52 (m, 4H), 1.83 (m, 1H), 1.80 (m, 1H), 1.53 (s, 9H), 1.39 (m,2H). EI-MS m/z: [M+4]⁺ 975.5.

Preparation of Compound 32c

Compound 32c was prepared from compound 1i and compound 32b by a similarmethod of preparing compound 25e in Example 35. EI-MS m/z: 1/2[M+H]⁺1508.8.

Example 48. Preparation of Compound 32d

Compound 32d was prepared from compound 1j and compound 32b by a similarmethod of preparing compound 25e in Example 35. EI-MS m/z: 1/2[M+H]⁺1522.8.

Example 49. Preparation of Compound 33e

Preparation of Compound 33a

DIPEA (1.98 mL, 11.37 mmol) and HBTU (2.15 g, 5.68 mmol) were added to astirred mixture of compound 24e (1.57 g, 3.79 mmol) and compound 6b(1.30 g, 3.15 mmol) in DMF (37 mL). After stirring at room temperaturefor 14 hours under N₂, the reaction mixture was poured into H₂O (40 mL)and extracted with EtOAc (3×40 mL). The combined organic layers werewashed with 1 N aq. HCl (40 mL), saturated aq. NaHCO₃ (40 mL) and brine(40 mL) sequentially, and dried over anhydrous Na₂SO₄. After filtrationand concentration, the residue was purified by column chromatography,which produced the compound 33a (2.2 g, 88%). ¹H-NMR (400 MHz, DMSO-d₆)δ 9.99 (s, 1H), 8.19 (d, 1H), 7.79 (t, 1H), 7.47 (d, 1H), 7.34-7.31 (m,5H), 7.24 (t, 1H), 5.01 (d, 4H), 4.55 (q, 1H), 3.91 (q, 1H), 3.79 (t,2H), 3.55-3.48 (m, 9H), 3.24-3.11 (m, 2H), 2.75-2.54 (m, 2H), 1.57-1.49(m, 2H), 1.38 (s, 9H), 1.25 (m, 2H). EI-MS m/z: [M+H]⁺ 790.47, [M+Na]⁺812.4.

Preparation of Compound 33b

To a stirred mixture of compound 33a (2.2 g, 2.78 mmol) and Pd/C (10 wt.%, 400 mg) in MeOH (60 mL) at 0° C. was added HCl (4 N in 1,4-dioxane,1.39 mL, 5.56 mmol). After stirring at room temperature for 3 hoursunder hydrogen, the reaction mixture was filtered through a celite padand washed with MeOH (40 mL). The filtrate was concentrated to producethe compound 33b (1.67 g, 99%), which was used without furtherpurification. ¹H-NMR (400 MHz, DMSO-d₆) δ 9.98 (s, 1H), 8.87 (d, 1H),8.28 (bs, 3H), 8.12 (1H), 7.96 (bs, 3H), 4.51 (q, 1H), 3.77 (t, 2H),3.72 (bs, 1H), 3.57 (s, 3H), 3.52-3.47 (m, 7H), 3.12 (s, 3H), 2.76-2.61(m, 4H) 1.71 (q, 2H), 1.55 (q, 2H) 1.36 (s, 9H). EI-MS m/z: [M+H]⁺522.4, [M+Na]⁺ 544.3.

Preparation of Compound 33c

DIPEA (1.95 mL, 11.23 mmol) and HBTU (3.19 g, 8.42 mmol) were added to astirred mixture of compound 25b (1.98 g, 6.17 mmol) and compound 33b(1.67 g, 2.80 mmol) in DMF (20 mL). After stirring at room temperaturefor 14 hours under N₂, the reaction mixture was concentrated andpurified by column chromatography, which produced the compound 33c (2 g,63%). ¹H-NMR (400 MHz, DMSO-d₆) δ 9.97 (s, 1H), 8.28 (d, 1H), 7.82 (t,1H), 7.65 (s, 1H), 7.64 (s, 1H), 4.54 (q, 1H), 4.25 (q, 1H), 3.91 (s,2H), 3.84 (s, 2H), 3.80 (t, 2H), 3.60-3.49 (m, 48H), 3.26-3.12 (m, 3H),3.07 (q, 2H), 2.75-2.54 (m, 2H), 1.65-1.55 (m, 2H), 1.39 (s, 10H), 1.21(m, 3H). EI-MS m/z: [M+H]⁺ 1128.8, [M+Na]⁺ 1150.7.

Preparation of Compound 33d

To a stirred mixture of compound 33c (1 g, 0.88 mmol) and Pd/C (10 wt.%, 200 mg) in MeOH (20 mL) at 0° C. was added HCl (4 N in 1,4-dioxane,0.44 mL, 0.88 mmol). After stirring at room temperature for 3 hoursunder hydrogen, the reaction mixture was filtered through a celite padand washed with MeOH (20 mL). The filtrate was concentrated to producethe compound 33d (936 mg, 92%), which was used without furtherpurification. ¹H-NMR (400 MHz, DMSO-d₆) δ 9.98 (s 1H), 8.30 (d, 1H),7.70 (t, 2H), 4.54 (q, 1H), 4.26 (q, 1H), 3.93 (s, 2H), 3.85 (s, 2H),3.80 (t, 2H), 3.61-3.49 (m, 46H), 3.22-3.12 (m, 4H), 3.06 (q, 2H), 2.97(q, 4H), 2.76-2.54 (m, 2H), 1.64-1.55 (m, 2H), 1.39 (s, 10H), 1.26 (m,3H). EI-MS m/z: [M+H]⁺ 1076.8.

Preparation of Compound 33e

Compound 33e was prepared from compound 1i and compound 33d by a similarmethod of preparing compound 25e in Example 35. EI-MS m/z: 1/2[M+H]⁺1552.2.

Example 50. Preparation of Compound 33f

Compound 33f was prepared from compound 1j and compound 33d by a similarmethod of preparing compound 25e in Example 35. EI-MS m/z: 1/2[M+H]⁺1566.4.

Example 51. Preparation of Compound 34e

Preparation of Compound 34a

DIPEA (0.8 mL, 4.56 mmol) and HBTU (1.3 g, 3.42 mmol) were added to astirred mixture of compound 24g (530 mg, 1.14 mmol) and Z-Asp(OMe)-OH(704 mg, 2.5 mmol) in DMF (5 mL). After stirring at room temperature for14 hours under N₂, the reaction mixture was poured into H₂O (50 mL) andextracted with EtOAc (3×30 mL). The combined organic layers were washedwith 1 N aq. HCl (40 mL), saturated aq. NaHCO₃ (40 mL) and brine (40 mL)sequentially, and dried over anhydrous Na₂SO₄. After filtration andconcentration, the residue was purified by column chromatography, whichproduced the compound 34a. (713 mg, 68%). ¹H-NMR (400 MHz, DMSO-d₆): δ9.97 (s, 1H), 7.88 (m, 3H), 7.64 (d, 2H), 7.51 (d, 2H), 7.35 (m, 10H),5.02 (m, 4H), 4.43-4.31 (m, 2H), 4.17 (m, 1H), 3.80 (t, 2H), 3.58-3.50(m, 12H), 3.41-3.16 (m, 6H), 2.98 (m, 2H), 2.79-2.67 (m, 3H), 2.57 (m,2H), 1.60-1.34 (m, 13H).

Preparation of Compound 34b

To a solution of compound 34a (530 mg, 0.58 mmol) in MeOH (5 mL) wasadded Pd/C (20 wt. %, 106 mg) and HCl (4 N in 1,4-dioxane, 0.29 mL, 1.16mmol). After stirring at room temperature for 3 hours under hydrogen,the reaction mixture was filtered through a celite pad and washed withMeOH (30 mL). The filtrate was concentrated to produce the compound 34b(420 mg, 100%), which was used without further purification. ¹H-NMR (400MHz, DMSO-d₆) δ 9.97 (s, 1H), 8.62 (d, 1H), 8.54 (s, 1H), 8.27 (m, 4H),7.02 (s, 1H), 4.17 (m, 2H), 4.02 (m, 1H), 3.76 (t, 2H), 3.61 (m, 4H),3.51-3.11 (m, 12H), 3.09-2.77 (m, 8H), 1.60-1.24 (m, 13H). EI-MS m/z:[M+H]⁺ 651.5.

Preparation of Compound 34c

DIPEA (0.4 mL, 2.32 mmol) and HBTU (660 mg, 1.74 mmol) were added to astirred mixture of compound 34b (420 mg, 0.58 mmol) and compound 25b(299 mg, 0.93 mmol) in DMF (5 mL). After stirring at room temperaturefor 14 hours under N₂, the reaction mixture was poured into H₂O (30 mL)and extracted with EtOAc (3×30 mL). The combined organic layers werewashed with 1 N aq. HCl (20 mL), saturated aq. NaHCO₃ (20 mL) and brine(20 mL) sequentially, and dried over anhydrous Na₂SO₄. After filtrationand concentration, the residue was purified by column chromatography,which produced the compound 34c (466 mg, 70.8%). ¹H-NMR (400 MHz,CDCl₃): δ 8.28 (s, 1H), 7.78 (q, 1H), 7.31 (d, 1H), 7.71 (s, 1H), 6.94(s, 1H), 4.85 (m, 2H), 4.35 (m, 1H), 4.07-4.03 (m, 6H), 3.75-3.41 (m,56H), 3.23 (q, 2H), 2.92-2.84 (m, 4H), 1.91-1.32 (m, 15H). EI-MS m/z:[M+2H]⁺ 1158.1.

Preparation of Compound 34d

To a stirred mixture of compound 34c (260 mg, 0.21 mmol) and Pd/C (10wt. %, 52 mg) in MeOH (20 mL) at 0° C. was added HCl (4 N in1,4-dioxane, 0.10 mL, 0.41 mmol). After stirring at room temperature for2 hours under hydrogen, the reaction mixture was filtered through acelite pad and washed with MeOH (50 mL). The filtrate was concentratedto produce the compound 34d (249 mg, 100%), which was used withoutfurther purification. EI-MS m/z: [M+2H]⁺ 1206.1.

Preparation of Compound 34e

Compound 34e was prepared from compound 1i and compound 34d by a similarmethod of preparing compound 25e in Example 35. EI-MS m/z: 1/2[M+H]⁺1610.4.

Example 52. Preparation of Compound 34f

Compound 34f was prepared from compound 1j and compound 34d by a similarmethod of preparing compound 25e in Example 35. EI-MS m/z: 1/2[M+H]⁺1624.3.

Example 53. Preparation of Compound 35g

Preparation of Compound 35a

DIPEA (0.61 mL, 3.52 mmol) and HBTU (665 mg, 1.175 mmol) were added to astirring mixture of Fmoc-D-Glu(OtBu)-OH (500 mg, 1.17 mmol) and compound2d (424 mg, 1.404 mmol) in DMF (10 mL). After stirring at roomtemperature for 14 hours under N₂, the reaction mixture was poured intoH₂O (30 mL) and extracted with EtOAc (3×30 mL). The combined organiclayers were washed with 1 N aq. HCl (20 mL), saturated aq. NaHCO₃ (20mL) and brine (20 mL) sequentially, and dried over anhydrous Na₂SO₄.After filtration and concentration, the resulting residue was purifiedby column chromatography, which produced the compound 35a (708 mg, 89%).EI-MS m/z: [M+H]⁺ 672.7.

Preparation of Compound 35b

To a solution of compound 35a (708 mg, 1.04 mmol) in THF (8 mL) wasadded piperidine (2 mL) at room temperature. After stirring for 20minutes, the reaction mixture was concentrated under reduced pressure.The resulting residue was purified by column chromatography, whichproduced the compound 35b (400 mg, 85%). EI-MS m/z: [M+H]⁺ 450.1.

Preparation of Compound 35c

DIPEA (0.19 mL, 1.1 mmol) and HBTU (253 mg, 0.66 mmol) were added to astirring mixture of compound 28a (203 mg, 0.484 mmol) and compound 35b(200 mg, 0.44 mmol) in DMF (10 mL). After stirring at room temperaturefor 14 hours under N₂, the reaction mixture was poured into H₂O (30 mL)and extracted with EtOAc (3×30 mL). The combined organic layers werewashed with 1 N aq. HCl (10 mL), saturated aq. NaHCO₃ (10 mL) and brine(10 mL) sequentially, and dried over anhydrous Na₂SO₄. After filtrationand concentration, the residue was purified by column chromatography,which produced the compound 35c (235 mg, 63%). EI-MS m/z: [M+H]⁺ 847.0.

Preparation of Compound 35d

To a solution of compound 35c (235 mg, 0.277 mmol) in MeOH (15 mL) wasadded Pd/C (10 wt. %, 30 mg). After stirring at room temperature for 2hours under hydrogen, the reaction mixture was filtered through a celitepad and washed with MeOH (50 mL). The filtrate was concentrated toproduce the compound 35d (160 mg, 100%), which was used without furtherpurification. EI-MS m/z: [M+H]⁺ 578.7.

Preparation of Compound 35e

DIPEA (0.145 mL, 1.758 mmol) and HBTU (262 mg, 1.465 mmol) were added toa stirring mixture of compound 35d (160 mg, 0.276 mmol) and compound 25b(187 mg, 0.581 mmol) in DMF (3 mL). After stirring at room temperaturefor 14 hours under N₂, the reaction mixture was poured into H₂O (30 mL)and extracted with EtOAc (3×30 mL). The combined organic layers werewashed with 1 N aq. HCl (10 mL), saturated aq. NaHCO₃ (10 mL) and brine(10 mL) sequentially, and dried over anhydrous Na₂SO₄. After filtrationand concentration, the residue was purified by column chromatography,which produced the compound 35e (260 mg, 79%). EI-MS m/z: [M+H]⁺ 1185.4.

Preparation of Compound 35f

To a solution of compound 35e (70 mg, 0.059 mmol) in MeOH (5 mL) wasadded Pd/C (10 wt. %, 15 mg). After stirring at room temperature for 90minutes under hydrogen, the reaction mixture was filtered through acelite pad and washed with MeOH (30 mL). The filtrate was concentratedto produce the compound 35f (67 mg, 100%), which was used withoutfurther purification. EI-MS m/z: [M+H]⁺: 1133.3.

Preparation of Compound 35g

Compound 35g was prepared from compound 1i and compound 35f by a similarmethod of preparing compound 25e in Example 35. EI-MS m/z: 1/2[M+H]⁺1559.9.

Example 54. Preparation of Compound 36e

Preparation of Compound 36a

DIPEA (0.3 mL, 3.10 mmol) and HBTU (474 mg, 2.275 mmol) were added to astirring mixture of the Fmoc-D-Glu(OtBu)-OH (484 mg, 1.138 mmol) andcompound 28b (223 mg, 0.569 mmol) in DMF (7 mL). After stirring at roomtemperature for 14 hours under N₂, the reaction mixture was poured intoH₂O (30 mL) and extracted with EtOAc (3×30 mL). The combined organiclayers were washed with 1 N aq. HCl (20 mL), saturated aq. NaHCO₃ (20mL) and brine (20 mL), and dried over anhydrous Na₂SO₄. After filtrationand concentration, the residue was purified by column chromatography,which produced the compound 36a (593 mg, 86%). EI-MS m/z: [M+H]⁺ 1208.3.

Preparation of Compound 36b

To a solution of compound 36a (593 mg, 0.49 mmol) in THF (8 mL) wasadded piperidine (1 mL) at room temperature. After stirring for 20minutes, the reaction mixture was concentrated under reduced pressure.The resulting residue was purified by column chromatography, whichproduced the compound 36b (166 mg, 44%). EI-MS m/z: [M+H]⁺ 763.9.

Preparation of Compound 36c

DIPEA (0.15 mL, 0.84 mmol) and HBTU (247 mg, 0.63 mmol) were added to astirred mixture of compound 36b (166 mg, 0.21 mmol) and compound 25b(147 mg, 0.441 mmol) in DMF (3 mL). After stirring at room temperaturefor 14 hours under N₂, the reaction mixture was poured into H₂O (30 mL)and extracted with EtOAc (3×30 mL). The combined organic layers werewashed with 1 N aq. HCl (10 mL), saturated aq. NaHCO₃ (10 mL) and brine(10 mL) sequentially, and dried over anhydrous Na₂SO₄. After filtrationand concentration, the residue was purified by column chromatography,which produced the compound 36c (195 mg, 68%). EI-MS m/z: [M+H]⁺ 1370.6.

Preparation of Compound 36d

To a solution of compound 36c (195 mg, 0.14 mmol) in MeOH (10 mL) wasadded Pd/C (10 wt. %, 30 mg). Then the reaction mixture was stirring atroom temperature for 90 minutes under hydrogen. The reaction mixture wasfiltered through a celite pad and washed with MeOH (30 mL). The filtratewas concentrated to produce the compound 36d (187 mg, 100%), which wasused without further purification. EI-MS m/z: [M+H]⁺ 1318.6.

Preparation of Compound 36e

Compound 36e was prepared from compound 1i and compound 36d by a similarmethod of preparing compound 25e in Example 35. EI-MS m/z: 1/2[M+H]⁺1624.4.

Example 55. Preparation of Compound 37d

Preparation of Compound 37a

DIPEA (0.083 mL, 0.71 mmol) and HBTU (136 mg, 0.36 mmol) were added to astirred mixture of propargyl amine (0.018 mL, 0.285 mmol) and compound1j (300 mg, 0.238 mmol) in DMF (3 mL). After stirring at roomtemperature for 14 hours under N₂, the reaction mixture was poured intoH₂O (10 mL) and extracted with EtOAc (3×10 mL). The combined organiclayers were washed with 1 N aq. HCl (10 mL), saturated aq. NaHCO₃ (10mL) and brine (10 mL) sequentially, and dried over anhydrous Na₂SO₄.After filtration and concentration, the resulting residue was purifiedby column chromatography, which produced the compound 37a (300 mg, 97%).EI-MS m/z: [M+H]⁺ 1294.0.

Preparation of Compound 37b

To a solution of compound 37a (300 mg, 0.24 mmol) in THF (2 mL) and MeOH(2 mL) was added LiOH monohydrate (50 mg, 1.20 mmol) in H₂O (2 mL) at 0°C. After stirring for 2 hours at 0° C., the reaction mixture wasneutralized using acetic acid and was concentrated under reducedpressure. Then the residue was dissolved in H₂O/DMSO (1.5 mL/1.5 mL) andpurified by HPLC. Pure fractions with the same retention time werecombined and concentrated to produce the compound 37b (165 mg, 60%).EI-MS m/z: [M+H]⁺ 1140.8.

Preparation of Compound 37c

CuSO₄.5H₂O (1 mg) and sodium ascorbate (2 mg) were added to a stirredmixture of compound 37b (50 mg, 0.042 mmol) and compound 25c (23 mg,0.02 mmol) in THF (2 mL) and H₂O (2 mL). The pH was adjusted to about 7by addition of 1 M aq. Na₂CO₃. After stirring at 20° C. for 1 hour, thereaction mixture was dissolved in H₂O/DMSO (1.5 mL/1.5 mL) and purifiedby HPLC. Pure fractions with the same retention time were combined andconcentrated to produce the compound 37c (32.4 mg, 48%). EI-MS m/z:1/2[M+H]⁺ 1638.2.

Preparation of Compound 37d

TFA (0.4 mL) was added to a solution of compound 37c (32.4 mg, 0.01mmol) in DCM (2 mL). After stirring at 0° C. for 2 hours, the solventand excess TFA were removed by N₂ flow. Then the residue was dissolvedin H₂O/MeCN (1 mL/1 mL) and purified by HPLC. Pure fractions with thesame retention time were combined and lyophilized to produce thecompound 37d (19.6 mg, 62%) as white solid. EI-MS m/z: 1/2[M+H]⁺ 1590.2.

Example 56. Preparation of Compound 38b

Preparation of Compound 38a

CuSO₄.5H₂O (1 mg) and sodium ascorbate (2 mg) were added to a stirredmixture of compound 37b (60 mg, 0.052 mmol) and compound 16b (14 mg,0.025 mmol) in THF (2 mL) and H₂O (2 mL). The pH was adjusted to about 7by addition of 1 M aq. Na₂CO₃. After stirring at 20° C. for 1 hour, thereaction mixture was dissolved in H₂O/DMSO (1.5 mL/1.5 mL) and purifiedby HPLC. Pure fractions with the same retention time were combined andconcentrated to produce the compound 38a (61 mg, 82%). EI-MS m/z:1/2[M+H]⁺ 1430.2.

Preparation of Compound 38b

TFA (0.4 mL) was added to a solution of compound 38a (59.8 mg, 0.02mmol) in DCM (2.0 mL). After stirring at 0° C. for 2 hours, the solventand excess TFA were removed by N₂ flow. Then the residue was dissolvedin H₂O/AN (1 mL/1 mL) and purified by HPLC. Pure fractions with the sameretention time were combined and lyophilized to produce the compound 38b(14.6 mg, 24%) as white solid. EI-MS m/z: 1/2[M+H]⁺ 1380.1.

Example 57. Preparation of Compound 38e

Preparation of Compound 38c

DIPEA (0.075 mL, 0.428 mmol) and HBTU (122 mg, 0.321 mmol) were added toa stirred mixture of propargyl amine (0.016 mL, 0.256 mmol) and compound1i (264 mg, 0.214 mmol) in DMF (3 mL). After stirring at roomtemperature for 14 hours under N₂, the reaction mixture was poured intoH₂O (10 mL) and extracted with EtOAc (3×10 mL). The combined organiclayers were washed with 1 N aq. HCl (10 mL), saturated aq. NaHCO₃ (10mL) and brine (10 mL) sequentially, and dried over anhydrous Na₂SO₄.After filtration and concentration, the residue was purified by columnchromatography, which produced the compound 38c (270 mg, 100%). EI-MSm/z: [M+H]⁺ 1266.2.

Preparation of Compound 38d

To a solution of compound 38c (270 mg, 0.213 mmol) in THF (2 mL) andMeOH (2 mL) was added LiOH monohydrate (36 mg, 0.853 mmol) in H₂O (2 mL)at 0° C. After stirring for 2 hours at 0° C., the reaction mixture wasneutralized using acetic acid and was concentrated under reducedpressure. Then the residue was dissolved in H₂O/DMSO (1.5 mL/1.5 mL) andpurified by HPLC. Pure fractions with the same retention time werecombined and concentrated to produce the compound 38d (168 mg, 70%).EI-MS m/z: [M+H]⁺ 1126.1.

Preparation of Compound 38e

Compound 38e was prepared from compound 38d and compound 16b by asimilar method of preparing compound 38b in Example 56. EI-MS m/z:1/2[M+H]⁺ 1366.2.

Example 58. Preparation of Compound 39e

Preparation of compound 39a

Compound 1g (27 mg, 0.039 mmol), compound 14j (45 mg, 0.039 mmol) andanhydrous HOBt (1 mg, 0.0078 mmol) were dissolved in DMF (2 mL) at 0° C.Then pyridine (0.2 mL) and DIPEA (0.014 mL, 0.078 mmol) were added.After stirring at 0° C. to room temperature for 24 hours under N₂, thereaction mixture was dissolved in DMSO (1 mL) and purified by HPLC,which produced the compound 39a (36 mg, 58%) as white solid. EI-MS m/z:[M+H]⁺ 1582.9, [M+Na]⁺ 1604.5.

Preparation of compound 39b

Compound 39a (35 mg, 0.022 mmol) and triphenylphosphine (1.5 mg, 0.005mmol) were dissolved in DCM (2 mL). Pyrrolidine (0.0025 mL, 0.026 mmol)and Pd(PPh₃)₄ (1.3 mg, 0.001 mmol) were added to the reaction mixture atroom temperature and then allowed to stir for 2 hours. The reactionmixture was diluted with H₂O (50 mL) and extracted with n-butanol (2×50mL). The combined organic layers were dried over anhydrous MgSO₄,evaporated under reduced pressure. The resulting residue was dissolvedin DMSO (1 mL) and purified by HPLC, which produced the compound 39b (34mg, crude) as white solid. EI-MS m/z: [M+H]⁺ : 1542.7.

Preparation of Compound 39c

DIPEA (0.0026 mL, 0.039 mmol) and PyBOP (4.7 mg, 0.023 mmol) were addedto a stirred mixture of compound 39b (15 mg, 0.009 mmol) and compound16c (2.0 mg, 0.0038 mmol) in DMF (0.3 mL). After stirring at roomtemperature for 13 hours under N₂, the reaction mixture was dissolved inDMSO (1.5 mL) and purified by HPLC, which produced the compound 39c (12mg, 35%) as white solid. EI-MS m/z: 1/2[M+H]⁺ 1788.5.

Preparation of Compound 39d

To a solution of compound 39c (12 mg, 0.0033 mmol) in MeOH (1 mL) wasadded LiOH monohydrate (1.4 mg, 0.033 mmol) in H₂O (1 mL) at 0° C. After2 hours at 0° C., the pH of the solution was adjusted with acetic acidto 4-5, and the reaction mixture was concentrated under reducedpressure. The resulting residue was dissolved in DMSO (1.5 mL) andpurified by HPLC, which produced the compound 39d (11 mg, 98%). EI-MSm/z: 1/2[M+H]⁺ 1648.6.

Preparation of Compound 39e

TFA (0.5 mL) was added to a stirred solution of compound 39d (11 mg,0.003 mmol) in DCM (3.0 mL) at 0° C. After 2 hours at 0° C., the solventand excess TFA were removed by N₂ flow. Then the residue was dissolvedin DMSO (1 mL) and purified by HPLC, which produced the compound 39e(1.2 mg, 11%) as white solid. EI-MS m/z: 1/2[M+H]⁺ 1598.3.

Example 59. Preparation of Compound 40c

Preparation of Compound 40a

DIPEA (0.004 mL, 0.021 mmol) and HBTU (5.0 mg, 0.013 mmol) were added toa stirred mixture of compound 39b (20 mg, 0.012 mmol) and compound 25d(5.0 mg, 0.005 mmol) in DMF (1.5 mL). After stirring at room temperaturefor 14 hours under N₂, the reaction mixture was dissolved in DMSO (1.0mL) and purified by HPLC, which produced the compound 40a (14.5 mg,30%). EI-MS m/z: 1/2[M+H]⁺ 1998.8.

Preparation of Compound 40b

To a solution of compound 40a (10 mg, 0.0025 mmol) in MeOH (1 mL) wasadded LiOH monohydrate (1.0 mg, 0.025 mmol) in H₂O (1 mL) at 0° C. After2 hours at 0° C., the reaction mixture was neutralized using acetic acidand concentrated under reduced pressure. Then the residue was dissolvedin DMSO (1.5 mL) and purified by HPLC, which produced the compound 40b(6.9 mg, 74%). EI-MS m/z: 1/2[M+H]⁺ 1858.3.

Preparation of Compound 40c

TFA (0.2 mL) was added to a stirred solution of compound 40b (6.9 mg,0.0018 mmol) in DCM (2.0 mL). After stirring at 0° C. for 2 hours, thesolvent and excess TFA were removed by N₂ flow. Then the residue wasdissolved in DMSO (1 mL) and purified by HPLC. Pure fractions with thesame retention time were combined and lyophilized to produce thecompound 40c (1.5 mg, 23%) as white solid. EI-MS m/z: 1/2[M+H]⁺ 1808.6.

Example 60. Preparation of Compound 41c

Preparation of Compound 41a

DIPEA (0.116 mL, 0.66 mmol) and PyBOP (127 mg, 0.24 mmol) were added toa stirred mixture of compound 16c (280 mg, 0.22 mmol) and compound 1j(587 mg, 1.10 mmol) in DMF (10 mL). After stirring at room temperaturefor 2 hours under N₂, the reaction mixture was diluted with H₂O (200 mL)and extracted with EtOAc (2×100 mL). The combined organic layers weredried over anhydrous MgSO₄, filtered and concentrated. The crude productwas purified by column chromatography to produce the compound 41a (250mg, 64%). EI-MS m/z: 1/2[M+H]⁺ 883.2, [M+H]⁺ 1766.

Preparation of Compound 41b

DIPEA (0.0017 mL, 0.0096 mmol) and PyBOP (2.0 mg, 0.0038 mmol) wereadded to a stirred mixture of compound 41a (5.7 mg, 0.0032 mmol) andcompound 39b (5.0 mg, 0.0032 mmol) in DMF (0.5 mL). After stirring atroom temperature for 3 hours under N₂, the reaction mixture wasdissolved in MeCN (1 mL) and purified by HPLC, which produced thecompound 41b (8.0 mg, 75%). EI-MS m/z: 1/2[M+H]⁺ 1645.

Preparation of Compound 41c

To a solution of compound 41b (8.0 mg, 0.0024 mmol) in MeOH (0.5 mL) wasadded LiOH monohydrate (1.2 mg, 0.028 mmol) in H₂O (0.1 mL) at 0° C.After 2 hours at 0° C., the reaction mixture was neutralized using 2 Naq. HCl solution and concentrated under reduced pressure. The resultingresidue was diluted with DCM (2 mL) and H₂O (3 drops). Then TFA (0.1 mL)was added at 0° C. After 2 hours at 0° C., the solvent and excess TFAwere removed by N₂ flow. Then the residue was dissolved in DMSO (1 mL)and purified by HPLC. Pure fractions with the same retention time werecombined and lyophilized to produce the compound 41c (3.1 mg, 44%) aswhite solid. EI-MS m/z: 1/2[M+H]⁺ 1448, 1/2[M+Na]⁺ 1459.

Example 61. Preparation of Compound 42d

Preparation of Compound 42a

DIPEA (0.026 mL, 0.23 mmol) and HBTU (22 mg, 0.06 mmol) were added to astirred mixture of compound 1j (60 mg, 0.048 mmol) and compound 25d (214mg, 0.19 mmol) in DMF (3 mL). After stirring at room temperature for 14hours under N₂, the reaction mixture was dissolved in DMSO (1.0 mL) andpurified by HPLC, which produced the compound 42a (64 mg, 58%). EI-MSm/z: [M+H]⁺ 2286.8.

Preparation of Compound 42b

DIPEA (0.011 mL, 0.06 mmol) and HBTU (14 mg, 0.036 mmol) were added to astirred mixture of compound 42a (68 mg, 0.03 mmol) and compound 39b (46mg, 0.03 mmol) in DMF (3 mL). After stirring at room temperature for 14hours under N₂, the reaction mixture was dissolved in DMSO (1.0 mL) andpurified by HPLC, which produced the compound 42b (60 mg, 52%). EI-MSm/z: 1/2[M+H]⁺ 1906.3.

Preparation of Compound 42c

To a solution of compound 42b (60 mg, 0.016 mmol) in MeOH (2 mL) wasadded LiOH monohydrate (5 mg, 0.126 mmol) in H₂O (2 mL) at 0° C. After 2hours at 0° C., the reaction mixture was neutralized using acetic acidand concentrated under reduced pressure. Then the residue was dissolvedin DMSO (1 mL) and purified by prep. HPLC, which produced the compound42c (37 mg, 65%). EI-MS m/z: 1/2[M+H]⁺ 1759.3.

Preparation of Compound 42d

TFA (0.3 mL) was added to a stirred solution of compound 42c (37 mg,0.01 mmol) in DCM (3 mL). After stirring at 0° C. for 2 hours, thesolvent and excess TFA were removed by N₂ flow. Then the residue wasdissolved in DMSO (1 mL) and purified by HPLC. Pure fractions with thesame retention time were combined and lyophilized to produce thecompound 42d (15 mg, 45%) as white solid. EI-MS m/z: 1/2[M+H]⁺ 1659.6.

Example 62. Preparation of Compound 43i

Preparation of Compound 43a

DIPEA (10.4 mL, 23.8 mmol) and HBTU (13.5 g, 35.7 mmol) were added to astirred mixture of H-Lys(z)-OMe hydrochloride (7.0 g, 23.8 mmol) andcompound 24e (9.86 mg, 23.8 mmol) in DMF (50 mL). After stirring at roomtemperature for 8 hours under N₂, the reaction mixture was diluted water(100 mL) and extracted with EtOAc (3×50 mL). The combined organic layerswere washed with 0.5 N aq. HCl (50 mL), saturated aq. NaHCO₃ (50 mL) andbrine (50 mL) sequentially, and dried over anhydrous Na₂SO₄. Afterfiltration and concentration under reduced pressure, the resulting crudeproduct was purified by column chromatography to produce the compound43a (9.3 g, 57%). ¹H-NMR (400 MHz, DMSO-d₆) δ 8.22 (d, 1H), 7.37-7.29(m, 15H), 7.22 (m, 2H), 5.00 (s, 6H), 4.18 (m, 1H), 4.00 (m, 1H), 3.59(s, 3H), 2.96 (m, 4H), 1.67-1.50 (m, 4H), 1.38-1.29 (m, 4H), 1.19-1.18(m, 4H). EI-MS m/z: [M+Na]⁺ 712.96.

Preparation of Compound 43b

To a solution of compound 43a (9.3 g, 13.5 mmol) in THF:MeOH:H₂O (60mL:30 mL:30 mL) was added LiOH monohydrate (1.13 g, 26.9 mmol) at 0° C.under N₂. After 2 hours, the reaction mixture was acidified with 1 N aq.HCl until pH 4, and extracted with EtOAc (3×100 mL). The combinedorganic layers were dried over anhydrous Na₂SO₄. Filtration andconcentration under reduced pressure provided compound 43b (9.1 g,crude), which was used without further purification. EI-MS m/z: [M+H]⁺677.48, 2[M+H]⁺ 1353.82.

Preparation of Compound 43c

DIPEA (1.47 mL, 8.44 mmol), HOBt (484 mg, 3.58 mmol) and EDC.HCl (809mg, 4.22 mmol) were added to a stirred mixture of compound 43b (2.5 g,3.71 mmol) and compound 3e (1.8 g, 3.38 mmol) in DMF (20 mL). Afterstirring at room temperature for 14 hours under N₂, the reaction mixturewas poured into water (50 mL) and extracted with EtOAc (3×50 mL). Thecombined organic layers were washed with 0.5 N aq. HCl (50 mL),saturated aq. NaHCO₃ (50 mL) and brine (50 mL) sequentially, and driedover anhydrous Na₂SO₄. After filtration and concentration under reducedpressure, the resulting residue was purified by column chromatography toproduce the compound 43c (2.3 g, 59%). EI-MS m/z: [M+H]⁺ 1155.92,[M+H-Boc]⁺ 1055.83.

Preparation of Compound 43d

To a stirred mixture of compound 43c (2.3 g, 1.99 mmol) and Pd/C (10 wt.%, 424 mg 3.98 mmol) in MeOH (200 mL) was added HCl (4 N in 1,4-dioxane,0.99 mL, 3.98 mmol). After stirring at room temperature for 2 hoursunder hydrogen, the reaction mixture was filtered through a celite padand washed with MeOH (100 mL). The filtrate was concentrated to producethe compound 43d (1.5 g, crude), which was used without furtherpurification. EI-MS m/z: [M+H]⁺ 753.29.

Preparation of Compound 43e

DIPEA (0.14 mL, 0.80 mmol), HOBt (59 mg, 0.43 mmol) and EDC.HCl (102 mg,0.53 mmol) were added to a stirred mixture of compound 43d (2.5 g, 3.71mmol) and compound 25b (150 g, 0.46 mmol) in DMF (5 mL). After stirringat room temperature for 14 hours under N₂, the reaction mixture waspoured into water (50 mL) and extracted with EtOAc (3×50 mL). Thecombined organic layers were washed with 1 N aq. HCl (30 mL), saturatedaq. NaHCO₃ (30 mL) and brine (30 mL) sequentially, and dried overanhydrous Na₂SO₄. After filtration and concentration under reducedpressure, the resulting crude product was purified by columnchromatography to produce the compound 43e (100 mg, 45%) as colorlessoil. EI-MS m/z: [M+Na]⁺ 1685.11, 1/2[M+H-Boc]⁺ 731.82.

Preparation of Compound 43f

To a stirred mixture of compound 43e (100 mg, 0.06 mmol) and Pd/C (10wt. %, 20 mg 0.192 mmol) in MeOH (20 mL) was added HCl (4 N in1,4-dioxane, 0.045 mL, 0.18 mmol). After stirring at room temperaturefor 2 hours under hydrogen, the reaction mixture was filtered through acelite pad and washed with MeOH (30 mL). The filtrate was concentratedto produce the compound 43f (95 mg) as brown foam, which was usedwithout further purification. EI-MS m/z: [M+H]⁺ 1586.30, 1/2[M+H]⁺793.02.

Preparation of Compound 43g

DIPEA (0.030 mL, 0.170 mmol) and HBTU (36 mg, 0.094 mmol) were added toa stirred mixture of compound 43f (45 mg, 0.028 mmol) and compound 1j(114 mg, 0.091 mmol) in DMF (3 mL). After stirring at room temperaturefor 16 hours under N₂, the reaction mixture was dissolved in H₂O/DMSO(1.5 mL/1.5 mL) and purified by HPLC. Pure fractions with the sameretention time were combined and concentrated to produce the compound43g (31 mg, 21%). EI-MS m/z: 1/3[M+H-Boc]⁺ 1705.74, 1/4[M+H−2Boc]⁺1254.79.

Preparation of Compound 43h

To a solution of compound 43g (31 mg, 0.006 mmol) in MeOH (1 mL) wasadded LiOH monohydrate (3.7 mg, 0.088 mmol) in H₂O (1 mL) at −20° C.After stirring for 2 hours at −20° C., the reaction mixture wasneutralized using acetic acid and concentrated under reduced pressure.Then the reaction mixture was dissolved in H₂O/DMSO (1.5 mL/1.5 mL) andpurified by HPLC, which produced the compound 43h (18 mg, 64%) as whitesolid. EI-MS m/z: 1/3[M+H-Boc]⁺ 1735.19, 1/4[M+H]⁺ 1301.95,1/5[M+H-Boc]⁺ 1021.71.

Preparation of Compound 43i

TFA (0.3 mL) was added to a stirred solution of compound 43h (18 mg,0.004 mmol) in DCM (1.0 mL) at 0° C. After stirring for 1 hour, thesolvent and excess TFA were removed by N₂ flow. Then the residue wasdissolved in H₂O/MeCN (1 mL/1 mL) and purified by HPLC. Pure fractionswith the same retention time were combined and lyophilized to producethe compound 43i (6 mg, 33%) as white solid. EI-MS m/z: 1/3[M+H]⁺1547.75, 1/4[M+H]⁺ 1161.14.

Example 63. Preparation of Compound 43j

Compound 43j was prepared from compound 1i and compound 43f by a similarmethod of preparing compound 43i in Example 62. EI-MS m/z: 1/3[M+H]⁺1532.37, 1/4[M+H]⁺ 1149.69.

Example 64. Preparation of Compound 44i

Preparation of Compound 44a

DIPEA (1.9 mL, 11.0 mmol) and HBTU (2.5 g, 6.64 mmol) were added to astirred mixture of compound H-Lys(z)-OMe hydrochloride (1.3 g, 4.43mmol) and compound 43b (3.0 g, 4.43 mmol) in DMF (30 mL). After stirringat room temperature for 14 hours under N₂, the reaction mixture wasdiluted water (100 mL) and extracted with EtOAc (3×50 mL). The combinedorganic layers were washed with 0.5 N aq. HCl (50 mL), saturated aq.NaHCO₃ (50 mL) and brine (50 mL), and dried over anhydrous Na₂SO₄. Afterfilteration and concentration under reduced pressure, the resultingresidue was purified by column chromatography to produce the compound44a (3.9 g, 93%). EI-MS m/z: [M+H]⁺953.42.

Preparation of Compound 44b

To a solution of compound 44a (2.1 g, 2.20 mmol) in THF:MeOH:H₂O (24mL:8 mL:8 mL) was added LiOH monohydrate (185 mg, 4.40 mmol) at roomtemperature under N₂. After 2 hours, the reaction mixture was acidifiedwith 1 N aq. HCl until pH 4, and extracted with EtOAc (3×50 mL). Thecombined organic layers were dried over anhydrous Na₂SO₄. Filtration andconcentration under reduced pressure provided compound 44b (2.0 g),which was used without further purification. EI-MS m/z: [M+H]⁺ 939.35,[M+Na]⁺ 961.37.

Preparation of Compound 44c

DIPEA (0.93 mL, 5.33 mmol) and HBTU (1.21 g, 3.20 mmol) were added to astirred mixture of compound 44b (2.0 g, 2.13 mmol) and compound 3e (1.14g, 2.13 mmol) in DMF (20 mL). After stirring at room temperature for 14hours under N₂, the reaction mixture was poured into water (50 mL) andextracted with EtOAc (3×50 mL). The combined organic layers were washedwith 0.5 N aq. HCl (50 mL), saturated aq. NaHCO₃ (50 mL) and brine (50mL) sequentially, and dried over anhydrous Na₂SO₄. After filtration andconcentration under reduced pressure, the resulting residue was purifiedby column chromatography to produce the compound 44c (2.60 g, 86%).EI-MS m/z: [M+H]⁺1418.44, [M+Na]⁺ 1440.39, [M+H-Boc]⁺ 1318.47.

Preparation of Compound 44d

To a stirred mixture of compound 44c (2.60 g, 1.83 mmol) and Pd/C (10wt. %, 781 mg 7.34 mmol) in MeOH (50 mL) was added HCl (4 N in1,4-dioxane, 0.9 mL, 3.67 mmol). And then the reaction was stirred atroom temperature for 2 hours under hydrogen. The reaction mixture wasfiltered through a celite pad and washed with MeOH (50 mL). The filtratewas concentrated to produce the compound 44d (1.73 g) as yellow form,which was used without further purification. EI-MS m/z: [M+H]⁺ 881.90.

Preparation of Compound 44e

DIPEA (1.58 mL, 9.08 mmol) and HBTU (2.58 g, 6.81 mmol) were added to astirred mixture of compound 44d (1.0 g, 1.13 mmol) and compound 25b(1.82 g, 5.67 mmol) in DMF (20 mL). After stirring at room temperaturefor 14 hours under N₂, the reaction mixture was diluted water (100 mL)and extracted with EtOAc (3×50 mL). The combined organic layers werewashed with 0.5 N HCl (50 mL), saturated aq. NaHCO₃ (50 mL) and brine(50 mL) sequentially, and dried over anhydrous Na₂SO₄. After filtrationand concentration under rescued pressure, the resulting residue waspurified by column chromatography to produce the compound 44e (848 mg,36%). EI-MS m/z: 1/2[M+H−2Boc]⁺ 947.63.

Preparation of Compound 44f

To a stirred mixture of compound 44e (848 mg, 0.40 mmol) and Pd/C (10wt. %, 172 mg 1.62 mmol) in MeOH (50 mL) was added HCl (4N in1,4-dioxane, 0.4 mL, 1.62 mmol). After stirring at room temperature for2 hours under hydrogen, the reaction mixture was filtered through acelite pad and washed with MeOH (50 mL). The filtrate was concentratedto produce the compound 44f (625 mg, crude), which was used withoutfurther purification. EI-MS m/z: 1/2[M+H]⁺ 996.40, 1/3[M+H]⁺ 664.59

Preparation of Compound 44g

DIPEA (0.067 mL, 0.386 mmol) and HBTU (110 mg, 0.289 mmol) were added toa stirred mixture of compound 44f (96 mg, 0.048 mmol) and compound 1j(303 mg, 0.24 mmol) in DMF (3 mL). After stirring at room temperaturefor 16 hours under N₂, the reaction mixture was dissolved in H₂O/DMSO(1.5 mL/1.5 mL) and purified by HPLC. Pure fractions with the sameretention time were combined and concentrated to produce the compound44g (67 mg, 20%). EI-MS m/z: 1/3[M+H]⁺ 2315.93, 1/4[M+H]⁺ 1737.60,1/5[M+H]⁺ 1390.37.

Preparation of Compound 44h

To a solution of the compound 44g (67 mg, 0.009 mmol) in MeOH (1 mL) wasadded LiOH monohydrate (8.1 mg, 0.192 mmol) in H₂O (1 mL) at −20° C.After stirring for 2 hours at −20° C., the reaction mixture wasneutralized using acetic acid and concentrated under reduced pressure.Then the reaction mixture was dissolved in H₂O/DMSO (1.5 mL/1.5 mL) andpurified by HPLC, which produced the compound 44h (27.9 mg, 45%) aswhite solid. EI-MS m/z: 1/3[M+H]⁺ 2110.24, 1/4[M+H]⁺ 1582.97,1/4[M+H-Boc]⁺ 1557.91.

Preparation of Compound 44i

TFA (0.3 mL) was added to a stirred solution of compound 44h (27.9 mg,0.004 mmol) in DCM (1.0 mL) at 0° C. After stirring for 1 hour, thesolvent and excess TFA were removed by N₂ flow. Then the residue wasdissolved in H₂O/MeCN (1 mL/1 mL) and purified by HPLC. Pure fractionswith the same retention time were combined and lyophilized to producethe compound 44i (13.6 mg, 50%) as white solid. EI-MS m/z: 1/3[M+H]⁺2043.49, 1/4[M+H]⁺ 1532.96, 1/5[M+H]⁺ 1226.62.

Example 65. Preparation of Compound 44j

Compound 44j was prepared from compound 1i and compound 44f by a similarmethod of preparing compound 44i in Example 64. EI-MS m/z: 1/3[M+H]⁺2025.37, 1/4[M+H]⁺ 1519.10, 1/5[M+H]⁺ 1215.60.

Example 66. Preparation of Compound 45k

Preparation of Compound L

D-Glucurono-6,3-lactone (25.0 g, 141.9 mmol) was dissolved in MeOH (250mL) at room temperature under nitrogen, and a solution of NaOH (141 mg)in MeOH (100 mL) was slowly added thereto. After stirring for 24 hours,the reaction mixture was concentrated under reduced pressure, and thenpyridine (66 mL) and acetic anhydride (71 mL) were added below 10° C.After stirring at room temperature for 4 hours, the reaction mixture wasconcentrated under reduced pressure and was subjected to columnchromatography, which produced the compound L (41.6 g, 77%). ¹H-NMR (600MHz, CDCl₃) δ 5.77 (d, J=7.8 Hz, 1H), 5.31 (t, J=9.6 Hz, 1H), 5.24 (t,J=9.6 Hz, 1H), 5.14 (m, 1H), 4.17 (d, J=9 Hz, 1H), 3.74 (s, 3H), 2.12(s, 3H), 2.04 (m, 9H).

Preparation of Compound M

Compound L (10.0 g, 26.6 mmol) was dissolved in HBr (33% in AcOH, 20 mL)at 0° C. under nitrogen. The reaction mixture was warmed to roomtemperature. After stirring for 2 hours, toluene (50 mL) was addedthereto, and the mixture was concentrated under reduced pressure. Theresulting residue was purified by column chromatography to produce thecompound M (10.9 g, 99%). ¹H-NMR (600 MHz, CDCl₃) δ 6.64 (d, J=3.6 Hz,1H), 5.61 (t, J=3.6 Hz, 1H), 5.24 (t, J=3.6 Hz, 1H), 4.85 (m, 1H), 4.58(d, d, J=10.2 Hz, 1H), 3.76 (s, 3H), 2.10 (s, 3H), 2.06 (s, 3H), 2.05(s, 3H).

Preparation of Compound 45a

3-Amino-1-propanol (3.0 g, 66.57 mmol) was dissolved in DCM (150 mL) at0° C. under nitrogen, and di-tert-butyl dicarbonate (16 g, 73.23 mmol)was added thereto. The obtained mixture was stirred at room temperaturefor 12 hours. After the reaction was completed, the solvent wasconcentrated under reduced pressure. The residue was subjected to columnchromatography, which produced the compound 45a (6.4 g, 92%). ¹H-NMR(400 MHz, CDCl₃) δ 4.78 (s, 1H), 3.65 (m, 2H), 3.30 (m, 2H), 2.90 (s,1H), 1.68 (m, 2H), 1.48 (s, 9H).

Preparation of Compound 45b

Compound 45a (6.04 g, 34.47 mmol) and triethylamine (14.4 mL, 103.4mmol) were dissolved in THF at 0° C. under nitrogen and then, slowlytreated with methanesulfonic anhydride (7.21 g, 41.36 mmol). Theobtained mixture was stirred at room temperature under nitrogen for 12hours. After the reaction was completed, the solvent was concentratedunder reduced pressure. The residue was subjected to columnchromatography, which produced the compound 45b (9.01 g, 98%). ¹H-NMR(400 MHz, CDCl₃) δ 4.73 (s, 1H), 4.30 (t, J=5.9 Hz, 2H), 3.31-3.24 (m,2H), 3.04 (s, 3H), 1.94 (t, J=6.1 Hz, 2H), 1.44 (s, 9H).

Preparation of Compound 45c

Compound 45b (3.0 g, 11.84 mmol) was dissolved in DMF (40 mL) at roomtemperature under nitrogen, and then treated with NaN₃ (924 mg, 14.21mmol), and the obtained mixture was stirred at 60° C. for 12 hours.After the reaction was completed, EtOAc (50 mL), distilled water (50mL), and 1 N aq. HCl (5 mL) were added thereto. The organic layer wasdried over anhydrous Na₂SO₄, filtered and concentrated under reducedpressure. The residue was subjected to column chromatography, whichproduced the compound 45c (2.3 g, 99%). ¹H-NMR (600 MHz, CDCl₃) δ 4.63(s, 1H), 3.36 (t, J=6.6 Hz, 2H), 3.24-3.18 (m, 2H), 1.80-1.75 (m, 2H),1.45 (s, 9H).

Preparation of Compound 45d

Compound 45c (3.8 g, 18.98 mmol) was dissolved in DCM (10 mL) at 0° C.under nitrogen, and then 4 M−HCl in dioxane (10 mL) was slowly addedthereto. After stirring for 12 hours, the reaction mixture wasconcentrated under reduced pressure, which produced the compound 45d(2.5 g, 99%). ¹H-NMR (600 MHz, DMSO-d₆) δ 8.06 (s, 3H), 3.47 (t, J=6.6Hz, 2H), 2.82 (t, J=7.2 Hz, 2H), 1.84-1.79 (m, 2H).

Preparation of Compound 45e

Compound 45d (58 mg, 0.42 mmol) and 5-formylsalicylic acid (100 mg, 0.60mmol) were dissolved in DMF (2 mL) at 0° C. under nitrogen, and thenDIPEA (0.2 mL, 1.20 mmol) and PyBop (375 mg, 0.72 mmol) were added tothe reaction mixture. After stirring at room temperature for 3 hours,EtOAc (30 mL) and distilled water (10 mL) were added thereto. Theorganic layer was dried over anhydrous Na₂SO₄, filtered and concentratedunder reduced pressure. The residue was subjected to columnchromatography, which afforded compound 45e (82 mg, 79%). ¹H-NMR (400MHz, CDCl₃) δ 13.39 (s, 1H), 9.87 (s, 1H), 8.29 (s, 1H), 7.89 (dd,J=1.6, 7.2 Hz. 1H), 7.60 (s, 1H), 7.10 (d, J=8.8 Hz, 1H), 3.63-3.57 (m,2H), 3.48 (t, J=6.4 Hz, 2H), 1.99-1.92 (m, 2H).

Preparation of Compound 45f

Compound 45e (78 mg, 0.31 mmol) and compound M (125 mg, 0.31 mmol) weredissolved in MeCN (3 mL) at room temperature under nitrogen, and thensilver oxide (291 mg, 1.26 mmol) and 4 Å molecular sieve (125 mg) wereadded thereto. After stirring at room temperature for 3 hours, themixture was celite-filtered, and the filtrate was concentrated underreduced pressure. The residue was subjected to column chromatography,which produced the compound 45f (160 mg, 90%). ¹H-NMR (400 MHz, CDCl₃) δ10.00 (s, 1H), 8.66 (d, J=2.4 Hz, 1H), 8.02 (dd, J=2.0, 6.4 Hz, 1H),7.46 (t, J=6.4 Hz, 1H), 7.14 (d, J=8.4 Hz, 1H), 5.48-5.33 (m, 4H), 4.28(d, J=8.8 Hz, 1H), 3.74 (s, 3H), 3.73-3.64 (m, 1H), 3.50-3.42 (m, 3H),2.09-2.07 (m, 9H), 2.00-1.92 (m, 2H).

Preparation of Compound 45g

Compound 45f (160 mg, 1.51 mmol) was dissolved in 2-propanol (0.4 mL)and chloroform (2 mL) at 0° C. under nitrogen, and then silica gel (2 g)and sodium borohydride (27 mg, 0.71 mmol) were added thereto. Afterstirring at 0° C. for 2 hours, the reactant was celite-filtered, and thefiltrate was concentrated under reduced pressure. The residue wassubjected to column chromatography, which produced the compound 45g (115mg, 71%). ¹H-NMR (600 MHz, CDCl₃) δ 8.06 (d, J=2.4 Hz, 1H), 7.50-7.44(m, 2H), 7.01 (d, J=9.0 Hz, 1H), 5.45-5.31 (m, 4H), 4.38 (s, 2H), 4.22(d, J=9.0 Hz, 1H), 3.74 (s, 3H), 3.67-3.61 (m, 1H), 3.46-3.41 (m, 3H),2.07-2.04 (m, 9H), 1.97-1.91 (m, 2H).

Preparation of Compound 45h

Compound 45g (100 mg, 0.18 mmol) was dissolved in DMF (1 mL) at 0° C.under nitrogen, and then bis(4-nitrophenyl)carbonate (110 mg, 0.35 mmol)and DIPEA (0.050 mL, 0.27 mmol) were added thereto. After stirring atroom temperature for 2 hours, EtOAc (30 mL) and distilled water (10 mL)were added thereto. The organic layer was dried over anhydrous Na₂SO₄and concentrated under reduced pressure. The residue was subjected tocolumn chromatography to produce the compound 45h (75 mg, 58%). ¹H-NMR(600 MHz, CDCl₃) δ 8.29-8.27 (m, 2H), 8.23 (d, J=2.4 Hz, 1H), 7.54 (dd,J=2.4, 6.6 Hz, 1H), 7.49 (t, J=6.4 Hz, 1H), 7.39-7.37 (m, 2H), 7.04 (d,J=8.4 Hz, 1H), 5.45-5.29 (m, 4H), 5.28 (s, 2H), 4.23 (d, J=9.0 Hz, 1H),3.75 (s, 3H), 3.68-3.64 (m, 1H), 3.46-3.42 (m, 3H), 2.08-2.05 (m, 9H),1.98-1.93 (m, 2H).

Preparation of Compound 45i

Compound 45h (50 mg, 0.068 mmol) was dissolved in DMF (0.8 mL) at roomtemperature under nitrogen, and then MMAF-OMe (51 mg, 0.068 mmol) wasadded thereto. The resulting mixture was treated with HOBT (2 mg, 0.013mmol), pyridine (0.24 mL), and DIPEA (0.012 mL, 0.068 mmol). Afterstirring at room temperature for 18 hours, EtOAc (20 mL) and distilledwater (10 mL) were added thereto. The organic layer was dried overanhydrous Na₂SO₄ and concentrated under reduced pressure. The residuewas subjected to column chromatography to produce the compound 45i (71mg, 78%). EI-MS m/z: [M+H]⁺ 1339.

Preparation of Compound 45j

Compound 45i (30 mg, 0.022 mmol) and phenylacetylene (3.7 μL, 0.033mmol) were dissolved in EtOH (0.2 mL) and water (30 μL) at roomtemperature under nitrogen, and then 0.1 M CuSO₄ aq. solution (30 μL)and 1.0 M sodium ascorbate aq. solution (30 μL) were added thereto. Theresulting mixture was treated with HOBT (2 mg, 0.013 mmol), pyridine(0.24 mL), and DIPEA (12 μL, 0.068 mmol). After stirring at roomtemperature for 5 hours, EtOAc (20 mL) and distilled water (5 mL) wereadded thereto. The organic layer was dried over anhydrous Na₂SO₄ andconcentrated under reduced pressure. The residue was subjected to columnchromatography to produce the compound 45j (26 mg, 81%). EI-MS m/z:[M+H]⁺ 1441.

Preparation of Compound 45k

Compound 45j (20 mg, 0.013 mmol) was dissolved in MeOH (0.2 mL) at 0° C.under nitrogen, and then LiOH.H₂O (6 mg, 0.14 mmol) in water (0.2 mL)was added thereto. After stirring at room temperature for 1 hour,chloroform (10 mL), MeOH (1 mL), distilled water (10 mL), and 0.5 N aq.HCl (1 mL) were added thereto. The organic layer was dried overanhydrous Na₂SO₄ and concentrated under reduced pressure. The residuewas subjected to column chromatography to produce the compound 45k (17mg, 87%). EI-MS m/z: [M+H]⁺ 1286.

Example 67. Preparation of Compound 46b

Preparation of Compound 46a 5-Formylsalicylic acid (1.0 g, 6.02 mmol)was dissolved in DMF (20 mL) at 0° C. under nitrogen, and thenN-bromosuccinimide (1.07 g, 6.11 mmol) was added thereto and the mixturewas stirred at 70° C. for 3 hours. After the reaction was completed,EtOAc (100 mL), 2 N aq. HCl solution (2 mL), and distilled water (100mL) were added thereto. The organic layer was dried over anhydrousNa₂SO₄ and concentrated under reduced pressure. The residue wassubjected to column chromatography to produce the compound 46a (1.2 g,84%). ¹H-NMR (400 MHz, DMSO-d₆) δ 9.64 (s, 1H), 8.19 (d, J=2.4 Hz, 1H),8.00 (d, J=2.0 Hz, 1H), 3.16 (s, 1H).

Preparation of Compound 46b

Compound 46b was prepared from compound 46a by a similar method ofpreparing compound 2h in Example 4. EI-MS m/z: [M+H]⁺ 1328.

Examples 68 to 70. Preparation of Compound 47a, Compound 48a, andCompound 49a

Compound N was prepared by a method disclosed in Korean Patent Laid-OpenPublication No. 10-2014-0035393.

Examples 68. Preparation of Compound 47a

Compound 2h (20 mg, 0.014 mmol) was dissolved in EtOH (0.7 mL) at roomtemperature under nitrogen, and then compound N (3.7 mg, 0.017 mmol) wasadded thereto, and the mixture was stirred at 45° C. for 2 hours. Afterthe reaction was completed, compound 47a (10.2 mg, 49%) was obtainedusing HPLC. EI-MS m/z: [M+H]⁺ 1441.

Examples 69 and 70. Preparation of Compound 48a and 49a

Compound 48a (Example 69) and compound 49a (Example 70) were prepared bya similar method of preparing compound 47a in Example 68. EI-MS ofcompound 48a m/z: [M+H]⁺ 1353. EI-MS of compound 49a m/z: [M+H]⁺ 1520.

Comparative Example 66. Preparation of Compound 50k

Preparation of Compound 50a

Ethyl 4-bromobutanoate (5.0 mL, 34.6 mmol) was dissolved in MeOH (7 5mL) at room temperature under nitrogen, and then NaN₃ (4.5 g, 69.2 mmol)in water (25 mL) was added thereto and stirred at 85° C. for 8 hours.After the reaction was completed, the solvent was concentrated underreduced pressure, and chloroform (300 mL) and distilled water (200 mL)were added thereto. The organic layer obtained as described above wasdried over anhydrous Na₂SO₄ and concentrated under reduced pressure. Theresidue was subjected to column chromatography to produce the compound50a (5.1 g, 94%). ¹H-NMR (600 MHz, CDCl₃) δ 4.15 (q, J=7.2 Hz, 2H), 3.36(t, J=7.2 Hz, 2H), 2.41 (t, J=7.2 Hz, 2H), 1.94-1.89 (m, 2H), 1.28 (t,J=8.4 Hz, 3H).

Preparation of Compound 50b

Compound 50a (2.0 g, 12.7 mmol) was dissolved in MeOH (32 mL) at 0° C.under nitrogen, and then KOH (3.56 g, 63.6 mmol) in water (26 mL) wasslowly added thereto. After stirring at room temperature for 6 hours,the solvent was concentrated under reduced pressure, and chloroform (300mL), 1 N aq. HCl (100 mL), and distilled water (100 mL) were addedthereto. The organic layer obtained as described above was dried overanhydrous Na₂SO₄ and concentrated under reduced pressure. The resultingresidue was subjected to column chromatography to produce the compound50b (1.28 g, 78%). ¹H-NMR (600 MHz, CDCl₃) δ 3.38 (t, J=7.2 Hz, 2H),2.48 (t, J=7.2 Hz, 2H), 1.95-1.90 (m, 2H).

Preparation of Compound 50c

Compound 50b (850 mg, 6.58 mmol) was dissolved in MeOH (10 mL) at 0° C.under nitrogen, and then oxalyl chloride (1.1 mL, 13.2 mmol) and DMF (1drop) were added thereto and stirred at room temperature for 6 hours.After the reaction was completed, the solvent was concentrated underreduced pressure to produce the compound 50c (965 mg), which was usedwithout further purification.

Preparation of Compound 50d 4-Hydroxy-3-nitrobenzoic acid (5.0 g, 27.3mmol) was dissolved in THF (120 mL) at 0° C. under nitrogen, and then 1M BH₃-THF complex (54.6 mL, 54.6 mmol) was added thereto and stirred atroom temperature for 20 hours. After the reaction was completed, EtOAc(200 mL), 0.5 N aq. HCl (20 mL), and distilled water (100 mL) were addedthereto. The organic layer obtained as described above was dried overanhydrous Na₂SO₄ and concentrated under reduced pressure. The resultingresidue was subjected to column chromatography to produce the compound50d (4.2 g, 91%). ¹H-NMR (600 MHz, CD₃OD) δ 8.06 (d, J=1.2 Hz, 1H), 7.59(dd, J=1.2, 7.8 Hz, 1H), 7.12 (d, J=8.4 Hz, 1H), 4.83 (s, 2H).

Preparation of Compound 50e

Compound 50d (937 mg, 5.54 mmol) was dissolved in MeCN (15 mL) at roomtemperature under nitrogen, and compound M (2.0 g, 5.04 mmol), silveroxide (4.66 g, 20.1 mmol), and 4 Å molecular sieve (2.0 g) were addedthereto, and stirred at room temperature for 14 hours. After thereaction was completed, the mixture was celite-filtered, and thefiltrate was concentrated under reduced pressure. The resulting residuewas subjected to column chromatography to produce the compound 50e (1.0g, 40%). ¹H-NMR (600 MHz, CDCl₃) δ 7.81 (d, J=1.8 Hz, 1H), 7.54 (dd,J=1.8, 6.6 Hz, 1H), 7.37 (d, J=8.4 Hz, 1H), 5.37-5.27 (m, 3H), 5.20 (d,J=6.6 Hz, 1H), 4.72 (d, J=6.0 Hz, 2H), 4.21 (d, J=9.0 Hz, 1H), 3.75 (s,3H), 2.12 (s, 3H), 2.06 (s, 3H), 2.05 (s, 3H), 2.04-2.02 (m, 1H).

Preparation of Compound 50f

Compound 50e (900 mg, 6.35 mmol) was dissolved in EtOAc (100 mL), andthen platinum (IV) oxide (84.2 mg, 0.370 mmol) was added thereto andstirred at room temperature under hydrogen for 3 hours. After thereaction was completed, the mixture was celite-filtered, and thefiltrate was concentrated under reduced pressure to produce the compound50f (700 mg, 83%), which was used without further purification.

Preparation of Compound 50g

Compound 50f (350 mg, 0.77 mmol) was dissolved in DCM (10 mL) at 0° C.under nitrogen, and then compound 50c (136 mg, 0.92 mmol) and DIPEA(0.27 mL, 1.54 mmol) were added thereto and stirred at room temperaturefor 20 minutes. After the reaction was completed, EtOAc (50 mL) anddistilled water (50 mL) were added thereto. The organic layer obtainedas described above was dried over anhydrous Na₂SO₄ and concentratedunder reduced pressure. The resulting residue was subjected to columnchromatography to produce the compound 50g (280 mg, 65%). ¹H-NMR (600MHz, CDCl₃) δ 8.37 (d, J=1.2 Hz, 1H), 8.00 (s, 1H), 7.07 (dd, J=1.8, 6.6Hz, 1H), 6.93 (d, J=8.4 Hz, 1H), 5.43-5.28 (m, 3H), 5.06 (d, J=7.8 Hz,1H), 4.63 (s, 2H), 4.19 (d, J=9.6 Hz, 1H), 3.76 (s, 3H), 3.44-3.41 (m,2H), 2.56 (t, J=7.8 Hz, 2H), 2.17-2.00 (m, 12H).

Preparation of Compound 50h

Compound 50g (250 mg, 0.44 mmol) was dissolved in DMF (4 mL) at 0° C.under nitrogen, and then bis(4-nitrophenyl)carbonate (270 mg, 0.88 mmol)and DIPEA (0.12 mL, 0.66 mmol) were added thereto, and stirred at roomtemperature for 1 hour. After the reaction was completed, EtOAc (50 mL)and distilled water (50 mL) were added thereto. The organic layerobtained as described above was dried over anhydrous Na₂SO₄ andconcentrated under reduced pressure. The resulting residue was subjectedto column chromatography to produce the compound 50h (290 mg, 90%).¹H-NMR (600 MHz, CDCl₃) δ 8.54 (d, J=1.8 Hz, 1H), 8.28-8.25 (m, 2H),8.02 (s, 1H), 7.40-7.36 (m, 2H), 7.11 (dd, J=1.8, 6.6 Hz, 1H), 6.96 (d,J=8.4 Hz, 1H), 5.44-5.29 (m, 3H), 5.23 (s, 2H), 5.10 (d, J=7.8 Hz, 1H),4.21 (d, J=9.6 Hz, 1H), 3.76 (s, 3H), 3.45-3.42 (m, 2H), 2.58 (t, J=7.2Hz, 2H), 2.11-2.00 (m, 12H).

Preparation of Compound 50i

Compound 50h (250 mg, 0.34 mmol) was dissolved in DMF (4 mL) at roomtemperature under nitrogen, and then MMAF-OMe (255 mg, 0.34 mmol) wasadded thereto. The resulting mixture was treated with HOBT (9 mg, 0.068mmol), pyridine (1.2 mL), and DIPEA (0.060 mL, 0.34 mmol). Afterstirring at room temperature for 2 days, EtOAc (50 mL), 2 N aq. HCl (5mL), and distilled water (50 mL) were added thereto. The organic layerobtained as described above was dried over anhydrous Na₂SO₄ andconcentrated under reduced pressure. The residue was subjected to columnchromatography to produce the compound 50i (340 mg, 74%). EI-MS m/z:[M+H]⁺ 1339.

Preparation of Compound 50j

Compound 50i (210 mg, 0.156 mmol) was dissolved in MeOH (2 mL) at 0° C.under nitrogen, and then LiOH.H₂O (66 mg, 1.56 mmol) in water (2 mL) wasadded thereto. After stirring at room temperature for 1.5 hours,chloroform (50 mL), MeOH (5 mL), distilled water (50 mL), and 0.5 N aq.HCl (5 mL) were added thereto. The organic layer obtained as describedabove was dried over anhydrous Na₂SO₄ and concentrated under reducedpressure. The resulting residue was subjected to column chromatographyto produce the compound 50j (107 mg, 57%). EI-MS m/z: [M+H]⁺ 1184.

Preparation of Compound 50k

Compound 50j (10 mg, 0.008 mmol) and phenylacetylene (0.92 μL, 0.008mmol) were dissolved in EtOH (0.15 mL) and water (10 μL) at roomtemperature under nitrogen, and then 0.1 M CuSO₄ aqueous solution (10μL) and 1.0 M sodium ascorbate aqueous solution (10 μL) were addedthereto. After stirring at room temperature for 5 hours, EtOAc (10 mL)and distilled water (5 mL) were added thereto. The organic layerobtained as described above was dried over anhydrous Na₂SO₄ andconcentrated under reduced pressure. The resulting residue was subjectedto column chromatography to produce the compound 50k (5 mg, 46%). EI-MSm/z: [M+H]⁺ 1286.

Example 71. Preparation of Compound 51h

Preparation of Compound 51a

Compound 51a was prepared from compound 7b by a method similar to methodof preparing compound 14h of Example 23. EI-MS m/z: [M+H]⁺ 1392.8,[M+H-Boc]⁺ 1292.7, [M+Na]⁺ 1414.8.

Preparation of Compound 51b

Compound 51a (1.8 g, 1.29 mmol), propargylamine (0.1 mL, 1.55 mmol) andanhydrous HOBt (35 mg, 0.25 mmol) were dissolved in DMF (5 mL) at 0° C.Then pyridine (0.2 mL) and DIPEA (0.45 mL, 2.59 mmol) were added. Afterstirring at room temperature for 24 hours under N₂, the reaction mixturewas diluted with H₂O (100 mL) and saturated aq. NH₄Cl solution (50 mL).After extraction with EtOAc (2×100 mL), the combined organic layers weredried over anhydrous MgSO₄, filtered, and concentrated. The residue waspurified by column chromatography to produce the compound 51b (1.15 g,68%). ¹H-NMR (400 MHz, CDCl₃) δ 8.01 (s, 1H), 7.48-7.31 (m, 2H), 7.02(d, J=8.4 Hz, 1H), 5.45-5.20 (m, 4H), 5.09 (s, 2H), 4.19 (d, J=9.2 Hz,1H), 4.10-4.05 (m, 2H), 3.97 (s, 2H), 3.85-3.45 (m, 49H), 2.24 (s, 1H),2.05 (s, 9H), 1.53 (s, 18H). EI-MS m/z: [M+Na]⁺1330.3.

Preparation of Compound 51c

To a solution of compound 51b (1.15 g, 0.879 mmol) in THF/MeOH (20 mL/20mL) was added LiOH monohydrate (151 mg, 3.603 mmol) in H₂O (20 mL) at 0°C. After 2 hours at 0° C., the reaction mixture was neutralized usingacetic acid and concentrated under reduced pressure. The resultingresidue was dissolved in DMSO (5 mL) and purified by prep. HPLC, whichproduced the compound 51c (600 mg, 60%). EI-MS m/z: [M+H]⁺ 1169.2.

Preparation of Compound 51d

DIPEA (0.92 mL, 5.30 mmol) and HBTU (1.0 g, 2.64 mmol) were added to astirred mixture of 4-azidobutanoic acid (228 mg, 1.76 mmol) andN-Me-Ala-OMe (298 mg, 1.94 mmol) in DMF (10 mL). After stirring at roomtemperature for 14 hours under N₂, the reaction mixture was diluted withH₂O (50 mL) and extracted with EtOAc (2×50 mL). The combined organiclayers were dried over anhydrous Na₂SO₄, filtered and concentrated. Theresulting residue was purified by column chromatography to yield thecompound 51d (310 mg, 77%). ¹H-NMR (400 MHz, CDCl₃) δ 5.22 (q, 1H), 3.71(s, 3H), 3.39 (t, J=6.6 Hz, 2H), 2.95 (s, 3H), 2.52-2.39 (m, 2H),1.98-1.92 (m, 2H), 1.41 (d, 3H).

Preparation of Compound 51e

To a solution of compound 51d (310 mg, 1.36 mmol) in MeOH (3 mL) wasadded LiOH monohydrate (114 mg, 2.72 mmol) in H₂O (3 mL) at −20° C.After stirring at 0° C. for 1 hour, the reaction mixture was dilutedwith H₂O/2 N aq. HCl solution (50 mL/2 mL) and extracted with Et₂O (2×30mL). The combined organic layers were dried over anhydrous Na₂SO₄.Filtration and concentration produced the compound 51e (246 mg), whichwas used without further purification. ¹H-NMR (400 MHz, CDCl₃) δ 5.15(q, 1H), 3.39 (t, J=6.6 Hz, 2H), 2.98 (s, 3H), 2.49-2.45 (m, 2H),1.98-1.92 (m, 2H), 1.41 (d, 3H).

Preparation of Compound 51f

To a solution of maytansinol (50 mg, 0.088 mmol) and compound 51e (113mg, 0.528 mmol) in DCM (6 mL) under N₂ was added a solution of DIC(0.087 mL, 0.557 mmol) in DCM (1.4 mL). After 1 minute, a solution ofZnCl₂ (1 M in Et₂O, 0.11 mL, 0.11 mmol) was added. After stirring atroom temperature for 2 hours, the reaction mixture was diluted withEtOAc (10 mL). The organic layer was washed with saturated aq. NaHCO₃ (4mL) and brine (2 mL), dried over anhydrous Na₂SO₄ and evaporated underreduced pressure. The resulting residue was purified by columnchromatography to yield a mixture of diastereomeric maytansinoidscompound 51f (50 mg, 74%). EI-MS m/z: [M+H]⁺ 761.7.

Preparation of Compound 51g

CuSO₄.5H₂O (2 mg) and sodium ascorbate (10 mg) were added to a stirringmixture of compound 51f (78 mg, 0.102 mmol) and compound 51c (132 mg,0.112 mmol) in DMSO (4 mL) and H₂O (1 mL). The pH was adjusted to about7 by addition of 1 M aq. Na₂CO₃. After stirring at 20° C. for 1 hour,the reaction mixture was dissolved in H₂O/DMSO (1.5 mL/1.5 mL) andpurified by HPLC. Pure fractions with the same retention time werecombined and concentrated to produce the compound 51g (72.1 mg, 37%).EI-MS m/z: [M+H]⁺ 1930.9, [M+H-Boc]⁺ 1830.9.

Preparation of Compound 51h

TFA (0.2 mL) was added to a stirring solution of compound 51g (72.1 mg,0.037 mmol) in DCM (1 mL). After stirring at 0° C. for 2 hours, thesolvent and excess TFA were removed by N₂ flow. Then the residue wasdissolved in H₂O/MeCN (1 mL/1 mL) and purified by HPLC. Pure fractionswith the same retention time were combined and lyophilized to producethe compound 51h (more polar isomer 17 mg and less polar isomer 6.0 mg,36%) as white solid. EI-MS m/z: [M+H]⁺ 1730.8.

Example 72. Preparation of Compound 52c

Preparation of Compound 52a

Taltobulin ethyl ester (TFA salt, 80 mg, 0.029 mmol), compound 14h (128mg, 0.0142 mmol) and anhydrous HOBt (3.5 mg, 0.026 mmol) were dissolvedin DMF (3 mL) at 0° C. Then pyridine (0.5 mL) and DIPEA (0.045 mL, 0.26mmol) were added. After stirring at room temperature for 24 hours underN₂, the reaction mixture was diluted with H₂O (10 mL) and extracted withEtOAc (2×10 mL). The combined organic layers were dried over anhydrousNa₂SO₄, filtered and concentrated. The resulting residue was purified bycolumn chromatography to yield the compound 52a (70 mg, 43%). EI-MS m/z:[M+H]⁺ 1258.6, [M+H-Boc]⁺ 1158.6.

Preparation of Compound 52b

To a solution of compound 52a (70 mg, 0.055 mmol) in MeOH (1.4 mL) wasadded LiOH monohydrate (11.7 mg, 0.275 mmol) in H₂O (1.4 mL) at −20° C.After 1 hour at 0° C., the pH of the solution was adjusted to 4-5 withacetic acid. The resulting solution was dissolved in DMSO (1 mL) andpurified by HPLC, which produced the compound 52b (4.5 mg, 8%) as whitesolid. EI-MS m/z: [M+H]⁺ 1090.4.

Preparation of Compound 52c

To a solution of compound 52b (4.5 mg, 0.0041 mmol) in DCM (1 mL) wasadded TFA (0.2 mL) at 0° C. After 2 hours at 0° C., the solvent andexcess TFA were removed by N₂ flow. Then the residue was purified byHPLC, which produced the compound 52c (2.4 mg, 59%) as white solid.EI-MS m/z: [M+H]⁺ 990.4.

Example 73. Preparation of Compound 53f

Preparation of Compound 53b

Compound 53a (300 mg, 0.31 mmol, Compound 53a was prepared by a methoddisclosed in patent WO2013/055987 A1), compound 15a (355 mg, 0.31 mmol)and anhydrous HOBt (10 mg, 0.06 mmol) were dissolved in DMF (0.5 mL) at0° C. Then pyridine (0.3 mL) and DIPEA (0.14 mL, 0.78 mmol) were added.After stirring at room temperature for 23 hours under N₂, the reactionmixture was diluted with H₂O/saturated aq. NH₄Cl solution (100 mL/50 mL)and extracted with EtOAc (2×100 mL). The combined organic layers weredried over anhydrous MgSO₄, filtered, and concentrated. The residue waspurified by column chromatography to produce the compound 53b (250 mg,41%). EI-MS m/z: [M+H]⁺ 1943.6, [M+Na]⁺ 1965.6.

Preparation of Compound 53c

To a solution of compound 53b (300 mg, 0.31 mmol) in THF/H₂O (2 mL/1 mL)was added acetic acid (3 mL) at 0° C. under N₂. After 22 hours, thereaction mixture was diluted with H₂O (100 mL) and extracted with EtOAc(2×100 mL). The combined organic layers were dried over anhydrousNa₂SO₄, filtered and concentrated. The resulting residue was purified bycolumn chromatography to yield the compound 53c (140 mg, 68%). EI-MSm/z: [M+H]⁺ 1713.6.

Preparation of Compound 53d

To a solution of compound 53c (120 mg, 0.07 mmol) in DCM (10 mL) wereadded pyridinium chlorochromate (158 mg, 0.42 mmol) and 4 Å molecularsieve (50 mg) at room temperature under N₂. After stirring for 18 hours,the reaction mixture was filtered through a celite pad and concentratedunder reduced pressure. The resulting compound 53d (95 mg, 75%) wasobtained as colorless oil, which was used without further purification.EI-MS m/z: [M+Na]⁺ 1732.8.

Preparation of Compound 53e

To a solution of compound 53d (95 mg, 0.056 mmol) in MeOH (1 mL) wasadded LiOH monohydrate (12 mg, 0.278 mmol) in H₂O (1 mL) at 0° C. After2 hours at 0° C., the reaction mixture was neutralized using acetic acidand concentrated under reduced pressure. The resulting residue wasdissolved in DMSO (1 mL) and purified by prep. HPLC, which produced thecompound 53e (6 mg, 7%). EI-MS m/z: [M+H]⁺ 1569.7.

Preparation of Compound 53f

TFA (0.2 mL) was added to a stirred solution of compound 53e (6 mg,0.004 mmol) in DCM (2 mL). After stirring at 0° C. for 2 hours, thesolvent and excess TFA were removed by N₂ flow. Then the residue wasdissolved in DMSO (1 mL) and purified by HPLC. Pure fractions with thesame retention time were combined and lyophilized to produce thecompound 53f (2.7 mg, 53%) as white solid. EI-MS m/z: [M+H]⁺ 1251.3.

Example 74. Preparation of Compound 54a

Compound 54a was prepared from compound 53a and compound 14h by asimilar method of preparing compound 53f in Example 73.

Example 75. Preparation of Compound 55a

Compound 55a was prepared from compound 53a and compound 51a by asimilar method of preparing compound 53f in Example 73. EI-MS m/z:[M+H]⁺ 1516.7, 1/2[M+H]⁺ 758.7.

Example 76. Preparation of Compound 56d

Preparation of Compound 56b

Compound 56a (HCl salt, 100 mg, 0.27 mmol, Compound 56a was prepared bya method disclosed in Curr. Med. Chem. 2009, 16, 1192-1213.), compound14h (242 mg, 0.27 mmol), and anhydrous HOBt (7.3 mg, 0.05 mmol) weredissolved in DMF (3 mL) at 0° C. Then pyridine (0.4 mL) and DIPEA (0.09mL, 0.60 mmol) were added. After stirring at room temperature for 16hours under N₂, the reaction mixture was diluted with saturated aq.NH₄Cl solution (10 mL) and extracted with EtOAc (2×20 mL). The combinedorganic layers were dried over anhydrous MgSO₄, filtered andconcentrated. The residue was purified by column chromatography toproduce the compound 56b (184 mg, 63%). EI-MS m/z: [M+H]⁺ 1091.9,[M+H-Boc]⁺ 991.7.

Preparation of Compound 56c

To a solution of compound 56b (90 mg, 0.08 mmol) in MeOH (2 mL) wasadded LiOH monohydrate (17 mg, 0.41 mmol) in H₂O (2 mL) at −20° C. Afterstirring for 2 hours at −20° C., the reaction mixture was neutralizedusing acetic acid and concentrated under reduced pressure. Then thereaction mixture was dissolved in H₂O/DMSO (1.5 mL/1.5 mL) and purifiedby HPLC, which produced the compound 56c (35 mg, 45%) as yellow solid.EI-MS m/z: [M+H]⁺ 951.7, [M+H-Boc]⁺ 851.5.

Preparation of Compound 56d

TFA (0.3 mL) was added to a stirred solution of compound 56c (35 mg,0.04 mmol) in DCM (2.0 mL) at 0° C. After stirring for 1 hour, thesolvent and excess TFA were removed by N₂ flow. Then the residue wasdissolved in H₂O/MeCN (1 mL/1 mL) and purified by HPLC. Pure fractionswith the same retention time were combined and lyophilized to producethe compound 56d (24.9 mg, 68%) as yellow solid. EI-MS m/z: [M+H]⁺851.6.

Example 77. Preparation of Compound 57a

Compound 57a was prepared from compound 56a and compound 15a by asimilar method of preparing compound 56d in Example 76. EI-MS m/z:[M+H]⁺ 983.3.

Example 78. ADC2 Synthesis

Example 79. ADC2 Synthesis

Example 80. ADC86 Synthesis

Example 81. ADC86 Synthesis

Example 82. ADC4 Synthesis

Example 83. ADC4 Synthesis

Example 84. ADC75 Synthesis

Example 85. ADC75 Synthesis

Experimental Example 1. Responsiveness Comparison Test with Respect toβ-Glucuronidase

In order to compare responsiveness of Compound 45k of Example 66 andCompound 50k of Comparative Example 66 to β-glucuronidase with eachother, comparison test was performed as follows.

Compound 45k of Example 66 and Compound 50k of Comparative Example 66were each prepared as 500 μM and 50 μM DMSO stock solutions. Reactionsolutions in which 880 μL of phosphate buffer saline (PBS) solution and100 μL of Compound 45k and Compound 50k stock solutions were mixed witheach other, respectively, were prepared (final concentrations thereofwere 50 μM and 5 μM, respectively). After 20 μL of E. coli(3-glucuronidase enzyme (1 mg/ml, Sigma: E.C.3.2.1.31 Type IX-A; 1 mg/mLin PBS; 3.6 μg, 13 μmol) was added to the reaction solutions, reactionswere initiated in a constant temperature water bath at 37° C. 100 μL ofthe mixed solutions were dispensed at 0 min, 25 min, 60 min, and 90 min,respectively, and 200 μL of acetonitrile was added thereto. MMAFreleased from each of the supernatants obtained by performingcentrifugation (4° C., 15 min, 14000 rpm) on the mixture samples wasquantitatively analyzed using LC-MS/MS (the experiment was performed bya method similar to a method disclosed in U.S. Pat. No. 8,568,728,hereby incorporated by reference).

The test results were illustrated in FIG. 2, and it was confirmed fromFIG. 2 that MMAF was significantly rapidly released from each Compound45k of Example 66 and Compound 50k of Comparative Example 66 through a1,6-elimination reaction after enzyme reactions by β-glucuronidase (U.S.Pat. No. 8,568,728, hereby incorporated by reference).

Experimental Example 2. Plasma Stability Comparison Test Linker Toxin

The plasma stability of Compound 45k of Example 66 and Compound 50k ofComparative Example 66 were compared.

10 μL of Compound 45k or 50k was dissolved in DMSO at 5 mM, and eachcomposition was mixed with 990 μL of mouse plasma, thereby preparing 50μM samples, for assessing plasma stability. The plasma/compoundsolutions were incubated at 37° C. for 7 days. During the 6-dayincubation, 100 μL aliquots were taken at 0, 1, 2, and 7 days and mixedwith 200 μL of acetonitrile containing an internal standard formonitoring plasma protein precipitation. Supernatants were obtained bycentrifuging the acetonitrile/plasma samples (4° C., 15 min, 14000 rpm),and the amount of each compound and product was quantified by performingLC-MS/MS on the supernatants. (The experiment was performed usingsimilar to those disclosed in J. Chromatography B, 780:451-457 (2002)).

Results obtained for Compound 45k of Example 66 and Compound 50k ofComparative Example 66 using LS-MS/MS are illustrated in FIG. 3 andTable 1. The stability of Compound 50k of Comparative Example 66 andstability of Compound 45k of Example 66 was 14% and 80% at 1 day,respectively. Thus, the stability of Compound 45k of Example 66 in mouseplasma was superior to Compound 50k of Comparative Example 66.

TABLE 1 Stability of Compound 45k and Compound 50k in mouse plasmaCompound 45k of Compound 50k of Example 66 Comparative Example 66 LinkerGlucuronide Glucuronide Plasma Stability 80% Stability 14% Stability(mouse plasma) (@7 days) (@1 day) Result Stable Unstable

The plasma stability of Compound 47a, 48a, and 49a were performed byusing the method mentioned above (FIG. 4-6).

Experimental Example 3. Preparation of Antibody-Drug Conjugate

Step 1. Method of Prenylated Antibody (prepared according to the methodof Korean Patent Laid-Open Publication No. 10-2014-0035393)

A prenylation reaction mixture of an antibody was prepared and reactedat 30° C. for 16 hours. The antibodies comprising the GGGGGGGCVIMsequence (“G7CVIM”) added to the c-terminus of each light chain wereused. The G7CVIM sequence was added at the C-terminus of heavy chain(ADC86-91) or both heavy and light chain (ADC75-77). The sources ofsequences of antibodies used were like following Table 2.

TABLE 2 The used antibody list for ADC preparation Target (Antibody)References HER2 (Herceptin ®) http://www.drugbank.ca/drugs/DB00002 EGFR(Erbitux ®) http://www.drugbank.ca/drugs/DB00002 CD19 (DI-B4) U.S. Pat.No. 8,691,952 B2 CD20 (Rituxan ®) http://www.drugbank.ca/drugs/DB00073EGFR wt & EGFRvIII US 2014/02555410 A9 (ABT806)

The reaction mixture was composed of a buffer solution (50 mM Tris-HCl(pH7.4), 5 mM MgCl₂, 10 μM ZnCl₂, 0.25 mM DTT) containing 24 μMantibody, 200 nM FTase (Calbiochem #344145), and 144 μM LCB14-0606(prepared in house according to the method of Korean Patent Laid-OpenPublication No. 10-2014-0035393, hereby incorporated by reference).After the reaction was completed, a prenylated antibody was purified byFPLC.

Step 2. Method of Preparing ADC

An oxime bond formation reaction mixture between the prenylated antibodyand linker-toxin was prepared by mixing 100 mM Na-acetate buffer (pH4.5, 10% DMSO), 12 μM prenylated antibody, and 120 μM linker-toxin (inhouse) and gently stirred at 30° C. After incubating the reaction for 24hours, the antibody-drug conjugate was purified by desalting via FPLCand hydrophobic interaction chromatography-HPLC.

TABLE 3 List of anti-HER2 ADCs (DAR2) ADC# Comp'd # ADC1  2g ADC2  2hADC3  3f ADC4  3g ADC5  4f ADC6  4g ADC7  5e ADC8  5f ADC9  6e ADC10  7eADC11  8f ADC12  9j ADC13 10c ADC14 10d ADC15 11j ADC16 11k ADC17 12cADC18 12d ADC19 13e ADC20 13f ADC86  2h ADC87  2g

TABLE 4 List of anti-HER2 ADCs (DAR4) ADC# Comp'd # ADC23 16f ADC24 16gADC25 17d ADC26 18c ADC27 19c ADC28 20q ADC29 21i ADC30 22h ADC31 23hADC32 24l ADC33 25e ADC34 25f ADC35 26e ADC36 27e ADC37 28d ADC38 28eADC39 29j ADC40 29k ADC41 30b ADC42 30c ADC43 31f ADC44 31g ADC45 32cADC46 32d ADC47 33e ADC48 33f ADC49 34e ADC50 34f ADC51 35g ADC52 36eADC53 37d ADC54 38b ADC55 38e ADC76  2h ADC88 16f ADC89 16g ADC90 25fADC91 25e

TABLE 5 List of anti-HER2 ADCs (DAR4<) ADC# Comp'd # <DAR6> ADC60 43iADC61 43j <DAR8> ADC62 44i ADC63 44j ADC77 16f

TABLE 6 List of anti-HER2 ADCs using amanitin as a payload DAR 2 DAR4ADC# Comp'd # ADC# Comp'd # ADC21 14m ADC56 39e ADC22 15b ADC57 40cADC58 41c ADC59 42d

TABLE 7 List of ADCs using antibodies targeting various proteins Target(Antibody) ADC# Comp'd # EGFR (Erbitux ®) ADC64  2h ADC65 25e ADC66 25fCD19 (DI-B4) ADC67  2h ADC68 25e ADC69 25f CD20 (Rituxan ®) ADC70  2hADC71 25e ADC72 25f EGFR wt & EGFRvIII ADC73  4g (ABT806) ADC74 25eADC75 25f

Experimental Example 4. Cytotoxicity of Anti-HER2 ADCs

Commercially available human breast cancer cell lines MCF-7 (HER2negative to normal), OE-19 (HER2 positive), NCI-N₈₇ (HER2 positive),SK-OV-3 (HER2 positive), JIMT-1 (HER2 positive), and SK-BR-3 (HER2positive) were used. The cell lines were cultured according torecommended specifications provided with the commercially available celllines.

Anti-proliferation activities of the antibodies, drugs, and conjugateswith regard to the cancer cell lines were measured. The cells wereplated in 96-well, tissue culture plates at 1×10⁴ cells per well. After24 hour incubation, the antibodies, drugs, and conjugates were added invarious concentrations. The number of viable cells after 72 hours werecounted using SRB assay. Absorbance was measured at 540 nm usingSpectraMax 190 (Molecular Devices, USA).

TABLE 8 IC₅₀ value of the different anti-HER2 ADCs (DAR2) Linker- NCI-Payload ADC toxin SK-BR-3 JIMT-1 OE-19 N87 SK-OV-3 MCF-7 MMAF ADC8  5f0.10 0.34 — — — >33.33 ADC2  2h 0.14 0.16 0.42 0.75 1.19 >33.33 ADC4  3g0.10 0.32 — 0.97 1.21 >33.33 ADC6  4g 0.03 0.38 — — — >33.33 ADC16 11k0.06 0.26 — — — >33.33 ADC14 10d 0.09 0.31 — — — >33.33 ADC18 12d 0.100.34 — — — >33.33 ADC20 13f 0.08 0.29 — — — >33.33 MMAE ADC7  5e 0.1013.44 — — — >33.33 ADC1  2g 0.47 275 — 0.69 241    >33.33 ADC3  3f 0.291.63 — 1.04 1.34 >33.33 ADC5  4f 0.15 0.97 — 0.40 1.04 >33.33 ADC9  6e0.10 >33.3 — — — >33.33 ADC15 11j 0.17 >33.3 — — — >33.33 ADC10  7e 0.120.64 — — — >33.33 ADC12  9j 0.20 2.01 — — — >33.33 ADC13 10c 0.19 1.09 —— — >33.33 ADC17 12c 0.03 0.11 — — — >33.33 ADC19 13e 0.13 0.38 — — —>33.33

TABLE 9 IC₅₀ value of the different PEG length-ADCs Test samples # ofPEG Linker- IC₅₀ (nM) Toxin DAR ADC unit toxin JIMT-1 MCF-7 MMAE 2 ADC71 5e >10.0 >10.0 ADC1 3 2g >10.0 >10.0 ADC3 6 3f >10.0 >10.0 ADC5 12 4f1.01 >10.0

TABLE 10 IC₅₀ value of MMAF ADCs with the different types of linkersLinker- SK- NCI- SK- ADC toxin BR-3 JIMT-1 N87 OV-3 MCF-7 ADC23 16f 0.050.12 0.35 0.44 >33.33 ADC34 25f 0.03 0.10 — — >33.33 ADC38 28e 0.03 0.05— — >33.33 ADC40 29k 0.02 0.06 — — >33.33 ADC42 30c 0.03 0.04 — — >33.33ADC44 31g 0.03 0.05 — — >33.33 ADC46 32d 0.04 0.08 — — >33.33 ADC48 33f0.03 0.08 — — >33.33 ADC50 34f 0.03 0.05 — — >33.33 ADC53 37d 0.03 0.05— — >33.33 ADC54 38b 0.05 0.12 — — >33.33

TABLE 11 IC₅₀ value of MMAE ADCs with the different types of linkersLinker- SK- NCI- SK- ADC toxin BR-3 JIMT-1 N87 OV-3 MCF-7 ADC24 16g 0.140.26 0.17 0.37 >33.33 ADC25 17d 0.03 0.10 0.36 0.37 >33.33 ADC26 18c0.08 0.14 — — >33.33 ADC27 19c 0.05 0.43 0.29 0.38 >33.33 ADC28 20q 0.030.12 0.36 0.36 >33.33 ADC29 21i 0.03 0.41 0.31 0.49 >33.33 ADC30 22h0.05 0.41 0.30 0.35 >33.33 ADC31 23h 0.14 0.24 — — >33.33 ADC32 24l 0.030.40 0.32 0.35 >33.33 ADC33 25e 0.04 0.23 0.27 0.33 >33.33 ADC35 26e0.13 0.19 — — >33.33 ADC36 27e 0.13 0.22 — — >33.33 ADC37 28d 0.04 0.07— — >33.33 ADC39 29j 0.03 0.11 — — >33.33 ADC41 30b 0.03 0.10 — — >33.33ADC43 31f 0.03 0.04 — — >33.33 ADC45 32c 0.03 0.08 — — >33.33 ADC47 33e0.04 0.07 — — >33.33 ADC49 34e 0.03 0.05 — — >33.33 ADC51 35g 0.04 0.240.34 — >33.33 ADC52 36e 0.07 0.40 0.37 — >33.33 ADC55 38e 0.04 0.07 — —>33.33

TABLE 12 IC₅₀ value of the hybrid ADCs Test samples IC₅₀ (nM) Linker-NCI- Toxin DAR ADC toxin SK-BR-3 JIMT-1 N87 SK-OV-3 MCF-7 MMAF 2 ADC2 2h 0.08 0.40 0.84 0.84 >33.33 4 ADC23 16f 0.05 0.10 0.25 0.36 >33.33Amanitin 2 ADC21 14m 0.09 >33.3 0.88 0.25 >33.33 MMAF & 4 ADC58 41c 0.060.73 0.95 0.64 >33.33 Amanitin 8 ADC78 41c 0.03 0.03 0.33 0.76 >33.33MMAF-OMe 0.12 0.07 0.49 0.78 0.60

The comparison of two different toxin conjugated ADCs and same toxinconjugated ADCs

TABLE 13 IC₅₀ value of the various DAR-ADCs Test samples Linker- IC₅₀(nM) DAR# ADC code toxin JIMT-1 MCF-7 DAR2 ADC2  2h 1.37 >33.33 DAR4ADC23 16f 0.21 >33.33 ADC34 25f 0.09 >33.33 ADC76  2h 0.27 >33.33 DAR8ADC62 44i 0.08 >33.33 ADC77 16f 0.02 >33.33

Experimental Example 5. Cytotoxicity of Erbitux (LC)-GlucuronideLinker-MMAF

A431 cells, which express high levels of EGFR, and MCF-7 cells, whichexpress low levels of EGFR, were plated at about 1000 cells per well ina 96-well plate in 100 μL of media. HCC-827 cells, which express anintermediate level of EGFR were plated at about 5000 cells per well in a96-well plate in 100 μL of media. The cells were incubated at 37° C. in5% CO₂ for 24 hours. Then, serial dilutions of monomethyl auristatinF-OMe (MMAF-OMe), Erbitux (LC)-G7CVIM, and the antibody drug conjugateADC64 comprising Erbitux (LC)-G7CVIM and MMAF were added to the cells atconcentrations of 100 to 0.00128 nM. The cells were incubated for 72hours and then fixed for 1 hour at 4° C. after adding 100 μL of ice-cold10% trichloroacetic acid to each well. Viable cells were counted usingSRB dye (Sulforhodamine B, Sigma 51402) and a Molecular DevicesSpectraMax 190 plate reader running Softmax Pro v5, monitoringabsorbance at 540 nm (Table 14)

Erbitux (LC)-G7CVIM had an IC50 greater than 100 nM for each cell line(A431, MCF-7, and HCC-827). MMAF-OMe had an IC50 of 1.81 nM againstMCF-7 cells, 1.99 nM against HCC-827 cells, and 1.11 nM against A431cells. The antibody-drug conjugate ADC64, 65, and 66 had an IC₅₀ ofgreater than 100 nM against MCF-7 cells, 0.47, 0.17, and 0.11 nM againstHCC-827 cells, respectively. ADC64 showed 1.3 nM against A431 cells,thus displaying superior specificity over MMAF-OMe and superior potencyover Erbitux (LC)-G7CVIM.

TABLE 14 IC₅₀ value of anti-EGFR mAb, Erbitux based ADCs ADC Cell-linesCode A431 HCC-827 MCF-7 ADC64 1.30 0.47 >33.33 ADC65 — 0.17 >33.33 ADC66— 0.11 >33.33

Experimental Example 6. Cytotoxicity of ABT-806 (LC)-Glucuronide LinkerMMAF′

Cytotoxicity of ABT-806 based ADCs were tested against patient derivedcell lines established Samsung Medical Center (Seoul, Republic ofKorea). The cells were maintained in Neurobasal®-A Media (Thermo FisherScientific) with supplement of L-glutamine (200 nM), bFGF (20 ng/mL),EGF (20 ng/mL), N₂ supplement, and B27 supplement. For the viabilitytest, cells were aliquoted to 96-well plate (5000 cells/well) andincubated at 37° C. in 5% CO₂ for 1 day before treatment. After ADCtreatment, cells were incubated for 72 hr. 100 μL of CellTiter-Glo®Reagent (Promega) was added to each well to analyze the cell viability.After 10 minutes incubation, luminescent signal was analyzed usingLuminometer.

DAR4 ADCs (ADC74, ADC75) had better potency than DAR2 ADC (ADC73) asexpected. Some patient's cells showed a little different sensitive topayload. 22 & 780 cells were more sensitive to MMAF over MMAE, 464 cellsvice versa.

TABLE 15 Cytotoxic activity of ABT-806 based ADCs against patientderived cell lines Patient Derived Cell-lines Test samples vIII352T1 780437 464 22 ABT806 ADC73 0.572 0.959 0.357 0.472 0.501 ADC75 0.104 0.2270.241 0.151 0.282 ADC74 0.170 0.425 0.253 0.069 0.489

Experimental Example 7. Cytotoxicity of Anti-CD19 ADCs

Ramos cells, which are human Burkitt's lymphoma cells, were seeded in a96-well plate at 20,000 cells/well in 100 μL of growth media. The cellswere incubated at 37° C. in 5% CO₂ for 1 day. Serial dilutions ofanti-CD19 antibodies DI-B4-(LC)-G7CVIM and ADCs from 33.33 nM to 5.1 pMin 100 μL media were added to the wells, and the cells were incubatedwith the antibody & ADCs for 72 hours. Cell viability was assessed usingWST-1 (TaKaRa MK400) and a Molecular Devices SpectraMax 190 plate readerrunning Softmax Pro v5, monitoring absorbance at 450 nm (Table 16).

The experiments in Ramos cells were performed in parallel withexperiments on K562 cells, human myelogenous leukemia cells that do notexpress CD19, as a negative control to assess any non-specificcytotoxicity.

ADC68 and ADC69 displayed an IC50 of 0.09 nM against Ramos cells, whichwas superior to unconjugated DI-B4 (Table 16). No antibody displayedcytotoxicity below 33.33 nM against the K562 control cells.

TABLE 16 Cytotoxic activity of anti-CD19 antibody based ADCs ADCCell-lines Code Ramos K562 ADC67 >33.33 >33.33 ADC68 0.09 >33.33 ADC690.09 >33.33

Experimental Example 8. Cytotoxicity of Rituxan Based ADCs

Ramos cells, which are human Burkitt's lymphoma cells, were seeded in a96-well plate at 20,000 cells/well in 100 μL of growth media. The cellswere incubated at 37° C. in 5% CO₂ for 1 day. Serial dilutions ofRituxan (LC)-G7CVIM and ADCs from 33.33 nM to 5.1 pM in 100 μL mediawere added to the wells, and the cells were incubated with the antibody& ADCs for 72 hours. Cell viability was assessed using WST-1 (TaKaRaMK400) and a Molecular Devices SpectraMax 190 plate reader runningSoftmax Pro v5, monitoring absorbance at 450 nm (Table 17).

The experiments in Ramos cells were performed in parallel withexperiments on K562 cells, human myelogenous leukemia cells that do notexpress CD20, as a negative control to assess any non-specificcytotoxicity.

ADC70, ADC71, and ADC72 displayed an IC₅₀ of 4.56 nM, 1.47 nM, and 1.78nM against Ramos cells respectively, which was superior to unconjugatedanti-CD20 antibody (Table 17). No antibody displayed cytotoxicity below33.33 nM against the K562 control cells.

TABLE 17 Cytotoxic activity of Rituxan-based ADCs ADC Cell-lines CodeRamos K562 ADC70 4.56 >33.33 ADC71 1.47 >33.33 ADC72 1.78 >33.33

Experimental Example 9. Differences in Beta Glucuronidase Susceptibility

ADCs in 0.06 M Na-acetate buffer (pH5.2) were aliquoted into the 1.5 mLmicro tube. The final concentration of ADC in the mixture was adjustedto 12 μM. 0.001 μg of human β-glucuronidase (R&D systems: 6144-GH-020)was added to each tube. Then, the mixtures were incubated at 37° C.water bath for 3 h. The reaction was terminated by the addition of coldPBS buffer (pH7.4) to the 15-fold dilution. The change of ADC-pattern bybeta-glucuronidase was analyzed by HIC-HPLC. The efficacy of enzymeactivity was visualized by % of remaining (FIG. 10)

The attribute to susceptibility seemed to be the Branch Unit (BR) oflinker-toxin part. When Lys was located in BR, the toxin release wasoccurred very efficiently. Amide and amine showed less susceptibilitythan Lys.

Experimental Example 10. Plasma Stability of ADCs

To compare plasma stability between ADC2 (Herceptin-LBG-MMAF, DAR2) andKadcyla, those ADCs were incubated in mouse and human plasma for 5seconds (0 h) or 96 hours (96 h), followed by SRB in vitro cytotoxicitytest using SK-BR3 cells for 72 hr. Plasma-incubated ADC2 retains potentcytotoxicity (no change in IC₅₀; 0.06 (0 h) and 0.07 nM (96 h) for MP,0.08 (0 h) and 0.08 nM (96 h) for HP) while plasma-incubated Kadcyladisplayed decreased cytotoxicity compared to 0 h Kadcyla (increase inIC₅₀; 0.26 (0 h) and 1.59 nM (96 h) for MP, 0.29 (0 h) and 4.21 nM (96h) for HP) (FIG. 11) To characterize the plasma stability of ADCs madeof various antibody, ADCs were incubated in human plasma for 5 seconds(0 h) or 168 hours (168 h), followed by SRB in vitro cytotoxicity testusing SK-BR3 cells for 72 hr. (Table 18-20, and FIG. 12)

TABLE 18 Plasma stability of Herceptin based ADCs (nM) Plasma incubationtime Test samples 0 h 168 h MMAF-OMe 0.48 N.D. Herceptin MMAF ADC2 0.090.15 ADC6 0.07 0.09 ADC8 0.11 0.18 ADC14 0.06 0.08 ADC16 0.05 0.07 ADC230.04 0.05 ADC34 0.03 0.04 ADC40 0.03 0.05 ADC46 0.03 0.03 ADC48 0.030.04 ADC62 0.02 0.02 MMAE ADC1 0.26 0.41 ADC3 0.20 0.32 ADC5 0.19 0.31ADC7 0.17 0.26 ADC12 0.51 0.79 ADC13 0.63 0.87 ADC15 0.52 0.70 ADC240.08 0.11 ADC25 0.07 0.12 ADC26 0.17 0.22 ADC27 0.10 0.14 ADC28 0.070.08 ADC29 0.06 0.08 ADC30 0.15 0.19 ADC31 0.08 0.12 ADC32 0.05 0.09ADC33 0.04 0.09 ADC35 0.11 0.21 ADC36 0.09 0.12 ADC39 0.12 0.17 ADC450.04 0.05 ADC47 0.04 0.05 ADC61 0.32 0.32

TABLE 19 Plasma stability of anti-CD19 antibody based ADCs (nM) Plasmaincubation Test samples 0 h 168 h MMAF-OMe 0.160 N.D. CD19 ADC68 0.0360.048 ADC69 0.047 0.135

TABLE 20 Plasma stability of anti-CD20 antibody based ADCs (nM) Plasmaincubation Test samples 0 h 168 h MMAF-OMe 0.160 N.D. CD20 ADC71 4.0014.134 ADC72 2.026 3.851

Experimental Example 11. Pharmacokinetics of Herceptin® and ADCs

Male Sprague Dawley rats were dosed intravenously with 3 mg/kg ofantibodies or the antibody-drug conjugates. Blood samples were taken atmultiple time points after dosing, chilled in ice water, and plasma wasisolated. Plasma was frozen at −80° C. until subsequent LC/MS/MSanalysis.

20 μL of each sample was mixed with 340 μL of PBS and 60 μL of protein Amagnetic beads and incubated for 2 hours at room temperature with gentleshaking. The beads were washed three times with PBS. Then, 25 μL of aninternal standard (isotope-labeled peptides at 10 μg/mL), 75 μL ofRapiGest SF (Waters), and 10 μL of dithiothreitol were added to thebeads. The mixture was shaken for 1 minute and then incubated for 1 hourat 60° C. 25 μL of iodoacetic acid was added to the mixture, the mixturewas shaken for 1 minute, and then incubated for 30 minutes at roomtemperature. 10 μL of sequencing grade modified trypsin (Promega) wasadded to the mixture, the mixture was shaken for 1 minute, and themixture was incubated overnight at 37° C. 15 μL of hydrochloric acid wasadded to the mixture, the mixture was shaken for 1 minute, and themixture was incubated for 30 minutes at 37° C. The mixture wascentrifuged at 5000×g for 10 minutes at 4° C. and the supernatant wastransferred into an HPLC vial.

The liquid chromatography-mass spectrometry system consisted of twoShimadzu LC-20AD pumps, a Shimadzu CBM-20A HPLC pump controller(Shimadzu Corporation, Columbia, Md., USA), a CTC HTS PAL autosampler(CEAP Technologies, Carrboro, N.C., USA) and a triple time of flight5600 mass spectrometer (Triple TOF MS) (AB Sciex, Foster City, Calif.,USA). The analytical column was a Phenomenex Kinetex XB-C18 column,2.1×30 (2.6 μm). HPLC was performed with a water/acetonitrile gradientand acidified with 0.1% formic acid. Injection volumes were 10 μL.Triple TOF MS, equipped with a Duospray™ ion source, was used tocomplete the high resolution experiment. The Triple TOF MS was operatedin the positive ion mode. High-purity nitrogen gas was used for thenebulizer/Duospray™ and curtain gases. The source temperature was set at500° C. with a curtain gas flow of 30 L/min. The ion spray voltage wasset at 5500 V, declustering potential was 145 V, and the collisionenergy was 38 V. The product ion mode was used as scan mode. Analyst® TFVersion 1.6 (AB Sciex) operated with Windows® (Microsoft) was used forinstrument control and data acquisition. Peak integrations wereperformed with MultiQuant® Version 2.1.1 (AB Sciex). Calculations wereperformed with MultiQuant® Version 2.1.1 for peak area ratios, standardcurve regressions, sample concentration values, and descriptivestatistics. The LC/MS/MS was calibrated using standard solutions atconcentrations of 0.1, 0.4, 1, 2, 5, 10, 20, 40, 80, and 100 μg/mL. Arepresentative PK profile was shown in FIG. 13. PK profile of ADC2(Herceptin (LC)-MMAF, DAR2) was a very similar with that of Herceptin.

Experimental Example 12. PEG (Connecting Unit) Combination Effect inBranched Linker-Toxin

To identify critical attributes that affect PK profile of ADC, differentlength and structure of connecting unit (PEG number and arrangement)were tested. Experiment for PK analysis was done as described inexperimental example 9. Although ADC23 (a DAR4 ADC) had more potentefficacy in vitro and in vivo than DAR2 ADCs, its PK profile was reducedin half life and AUC (FIG. 14). By replacing linker-toxin from 16f to25f (attaching additional connecting unit (3 PEGs) to after branch unit(BR), PK profile had been recovered as much as that of Herceptin (FIG.14). These effects were well reproduced in MMAE based ADCs, ADC24 &ADC33 (FIG. 15). Because MMAE is more hydrophobic than MMAF, ADCs basedon MMAE has worsened in PK profile as shown by ADC1 and ADC24. Addinglonger PEG unit was traditional application for extending half-life andAUC. However, simple elongation of PEG unit numbers from 3 to 12 didn'tshow big differences, when comparing ADC1 with ADC5. In the other hand,by replacing linear linker unit (compound 2g or 40 to branched one(compound 11j, ADC15), the PK profile was significantly improved (FIG.16), indicating that critical attribute for PK might not be a justsimple length, but the structure of connecting unit.

Experimental Example 13. Effects of Hydrophilic Connecting Unit in PK ofADC

Many payloads used for ADC have hydrophobic character, resulted in badPK property. To compensate the hydrophobicity, hydrophilic compoundswere tested as a part of connecting unit. Inserting hydrophiliccompounds such as Asp enhanced AUC and half-life of ADCs (FIG. 17, 18,19). In cases of DAR2, ADC with connecting unit including Asp showedhigher AUC than Herceptin (FIG. 17, 18). The compensate effect by polaramino acid, such as Asp or Glu, can be observed in ADCs with DAR4 (FIG.19, 20). ADC49 (2 Asp) and ADC52 (2 D-Glu) were superior to ADC47 (1Asp) and ADC51 (1D-Glu) respectively in AUC and half-life.

Experimental Example 14. In Vivo Efficacy

A frozen JIMT-1 cell stock was thawed and cultivated under the 37° C.,5% CO₂ condition. JIMT-1 cells of the best condition that the viabilitywas more than 95% were used for implantation. Cells of 5×10⁶ suspendedin 50 μL cold-saline were implanted into right hind leg of balb/c-nudemouse. 5 mice per group were used for the experiments. Tumor formationand growth were periodically monitored. Tumor volume was calculated bythe formulation; volume=(a²b)/2, “a” means short diameter and “b” meanslong diameter.

When the tumor volume reaches to about 200 mm³, mice having averagevalue were selected and grouped according to tumor volume. Then, micewere treated with PBS (vehicle control), or ADCs indicated in FIGS. 21and 22. Tumor size was determined 2 times a week in 3˜4 days intervalduring the experimental period. Tumor volumes measured from the firstday of administration to the end date were plotted for tumor growthcurve.

Representative ADCs were tested by single injection. In general, theADCs with branching unit (BR) containing Lys had better efficacy thanADCs with BR containing amide.

INCORPORATION BY REFERENCE

Each of the patents, published patent applications, and non-patentreferences cited herein are hereby incorporated by reference in theirentirety.

EQUIVALENTS

Those skilled in the art will recognize, or be able to ascertain usingno more than routine experimentation, many equivalents to the specificembodiments of the invention described herein. Such equivalents areintended to be encompassed by the following claims.

What is claimed:
 1. A ligand-active agent conjugate, comprising aligand, a branched linker, at least two cleavage groups, and at leasttwo active agents, wherein: the ligand is covalently coupled to thebranched linker; each cleavage group is covalently coupled to thebranched linker; each active agent is covalently coupled to one cleavagegroup; the branched linker comprises a peptide sequence of a pluralityof amino acids wherein the cleavage groups are covalently coupled to theside chains of the amino acids; and each cleavage group has a structureconsisting of formula I:

wherein: G is a sugar, sugar acid, or modified sugar; W is —C(O)—,—C(O)NR′—, —C(O)O—, —S(O)₂NR′-, —P(O)R″NR′—, —S(O)NR′—, or -PO₂NR′-, ineach case wherein the C, S, or P is directly bound to the phenyl ring,and the N or O is directly bound to the branched linker; R′ and R″ areeach independently hydrogen, (C₁-C₈)alkyl, mono- ordi-carboxyl(C₁-C₈)alkyl, (C₃-C₈)cycloalkyl, (C₁-C₈)alkoxy,(C₁-C₈)alkylthio, mono- or di-(C₁-C₈)alkylamino, (C₃-C₂₀)heteroaryl, or(C₆-C₂₀)aryl; each Z independently is hydrogen, (C₁-C₈)alkyl, or anelectron-withdrawing group; n is an integer from 1 to 3; m is 0 or 1; R₁and R₂ are each independently hydrogen, (C₁-C₈)alkyl, or(C₃-C₈)cycloalkyl, or R₁ and R₂ taken together with the carbon atom towhich they are attached form a (C₃-C₈)cycloalkyl ring;

is a connection to the branched linker; and

is a connection to the active agent.
 2. The conjugate of claim 1,wherein the ligand is an antibody.
 3. The conjugate of claim 1, whereinat least two of the active agents are covalently coupled via thecleavage groups to side chains of lysine, 5-hydroxylysine, 4-oxalysine,4-thialysine, 4-selenalysine, 4-thiahomolysine, 5,5-dimethyllysine,5,5-difluorolysine, trans-4-dehydrolysine, 2,6-diamino-4-hexynoic acid,cis-4-dehydrolysine, 6-N-methyllysine, diaminopimelic acid, ornithine,3-methylornithine, a-methylornithine, citrulline, and/or homocitrulline.4. The conjugate of claim 1, wherein the plurality of amino acidscomprises alanine, aspartate, asparagine, glutamate, glutamine, glycine,histidine, lysine, ornithine, proline, serine, or threonine.
 5. Theconjugate of claim 1, wherein: the branched linker is covalently boundto the ligand by a thioether bond; the ligand comprises a C-terminalamino acid motif that is recognized by an isoprenoid transferase; andthe thioether bond comprises a sulfur atom of a cysteine of theC-terminal amino acid motif.
 6. The conjugate of claim 5, wherein: theC-terminal amino acid motif is a sequence CYYX; C is cysteine; Y,independently for each occurrence, is an aliphatic amino acid; X,independently for each occurrence, is glutamine, glutamate, serine,cysteine, methionine, alanine, or leucine; and the thioether bond isformed from a sulfur atom of a cysteine of the C-terminal amino acidmotif.
 7. The conjugate of claim 5, wherein the thioether bond comprisesa carbon atom of at least one isoprenyl moiety.
 8. The conjugate ofclaim 1, wherein the branched linker further comprises a connection unitselected from the group consisting of—(CH₂)_(r)(V(CH₂)_(p))_(q)-,-((CH₂)_(p)V)_(q)-, -(CH₂)_(r)(V(CH₂)_(p))_(q)Y₁-,-((CH₂)_(p)V)_(q)(CH₂)_(r)-, -Y₁(((CH₂)_(p)V)_(q)- and-(CH₂)_(r)(V(CH₂)_(p))_(q)Y₁CH₂— wherein: r is an integer from 0 to 10;p is an integer from 1 to 10; q is an integer from 1 to 20; V and Y₁ areeach independently a single bond, —O—, —S—, —NR₂₁—, —C(O)NR₂₂—,—NR₂₃C(O)—, —NR₂₄SO₂—, or —SO₂NR₂₅—; and R₂₁ to R₂₅ are eachindependently hydrogen, (C₁-C₆)alkyl, (C₁-C₆)alkyl(C₆-C₂₀)aryl or(C₁-C₆)alkyl(C₃-C₂₀)heteroaryl.
 9. The conjugate of claim 1, wherein thebranched linker further comprises a binding unit, and the binding unithas a structure according to Formula A, B, C, or D:

wherein: L₁ is a single bond or alkylene having 1 to 30 carbon atoms;R₁₁ is hydrogen or alkyl having 1 to 10 carbon atoms; and L₂ is alkylenehaving 1 to 30 carbon atoms.
 10. The conjugate of claim 1, wherein eachbranched linker comprises a branching unit, and each active agent iscoupled to the branching unit through the cleavage group and a secondarylinker; and the branching unit is coupled to the peptide by a primarylinker.
 11. The conjugate of claim 10, wherein at least one branchingunit has the structure

wherein L₁₁, L₂₂, L₃₃ is each independently a bond or —C_(n)H_(2n)—where n is a integer of 1 to 30; wherein G₁, G₂, G₃ is eachindependently a bond,

wherein R₃₃ is hydrogen or C₁-C₃₀ alkyl; wherein R₄₄ is hydrogen orL₄-COOR₅₅, wherein L₄ is a bond or —C_(n)H_(2n)— wherein n is a integerof 1 to 10, and R₅₅ is hydrogen or C₁-C₃₀ alkyl.
 12. The conjugate ofclaim 1, wherein the ligand is selected from the group consisting ofmuromonab-CD3 abciximab, rituximab, daclizumab, palivizumab, infliximab,trastuzumab, etanercept, basiliximab, gemtuzumab, alemtuzumab,ibritumomab, adalimumab, alefacept, omalizumab, efalizumab, tositumomab,cetuximab, ABT-806, bevacizumab, natalizumab, ranibizumab, panitumumab,eculizumab, rilonacept, certolizumab, romiplostim, AMG-531, golimumab,ustekinumab, ABT-874, belatacept, belimumab, atacicept, an anti-CD20antibody, canakinumab, tocilizumab, atlizumab, mepolizumab, pertuzumab,HuMax CD20, tremelimumab, ticilimumab, ipilimumab, IDEC-114, inotuzumab,HuMax EGFR, aflibercept, HuMax-CD4, teplizumab, otelixizumab,catumaxomab, the anti-EpCAM antibody IGN101, adecatumomab, oregovomab,dinutuximab, girentuximab, denosumab, bapineuzumab, motavizumab,efumgumab, raxibacumab, LY2469298, and veltuzumab.
 13. The conjugate ofclaim 1, wherein: G is

R₃ is hydrogen or a carboxyl protecting group; and each R₄ isindependently hydrogen or a hydroxyl protecting group.
 14. The conjugateof claim 1, wherein W is —C(O)NR′—, where C is bonded to the phenyl ringand NR′ is bonded to the branched linker.
 15. A pharmaceuticalcomposition comprising the conjugate of claim 1 and a pharmaceuticallyacceptable excipient.
 16. A method of inhibiting cancer cell growth orkilling cancer cell(s) in a subject in need thereof comprisingadministering the conjugate of claim 1 to the subject, wherein theconjugate comprises a ligand which targets the cancer cell(s).
 17. Amethod for making the ligand-active agent conjugate of claim 1,comprising providing a biomolecule comprising a ligand, a branchedlinker, at least 2 cleavage groups, and a reactive group selected fromthe group consisting of a ketone and an aldehyde to react with a prodrugcomprising an active agent and an alkoxyamine; and the reaction producesan oxime, thereby covalently linking the biomolecule to the prodrug toproduce the conjugate of claim
 1. 18. The conjugate of claim 1, whereinZ is an amide, carboxylic acid, carboxylic acid ester, halogen, cyano,or nitro.
 19. The conjugate of claim 1, wherein the peptide sequence ofthe branched linker comprises 2 to 20 amino acids.
 20. The conjugate ofclam 63, wherein: the ligand is an antibody; G is

R₃ and each R₄ is hydrogen; W is —C(O)NR′—, where C(O) is bonded to thephenyl ring and NR′ is bonded to the branched linker; R′ is hydrogen; R₁and R₂ are each hydrogen; m is 1; Z is halogen, cyano, or nitro; theconjugate comprises two, three, or four active agents and cleavagegroups; the peptide sequence of the branched linker comprises 2 to 20amino acids selected from the group consisting of alanine, aspartate,asparagine, glutamate, glutamine, glycine, histidine, lysine, ornithine,proline, serine, and threonine; and the branched linker furthercomprises a binding unit with a structure according to Formula A, B, C,or D:

wherein: L₁ is a single bond or alkylene having 1 to 30 carbon atoms;R₁₁ is hydrogen or alkyl having 1 to 10 carbon atoms; and L₂ is alkylenehaving 1 to 30 carbon atoms.
 21. The conjugate of claim 1, wherein theconjugate comprises:

and MMAE is monomethyl auristatin E; MMAF is monomethyl auristatin F;and

is a connection to the remaining portion of the branched linker whichconnects to the ligand.